Cationic sulfonamide amino lipids and amphiphilic zwitterionic amino lipids

ABSTRACT

The present disclosure provides one or more amino lipids such as an amino lipids containing a sulfonic acid or sulfonic acid derivative of the formulas: 
     
       
         
         
             
             
         
       
     
     wherein the variables are as defined herein. These amino lipids may be used in compositions with one or more helper lipids and a nucleic acid therapeutic agent. These compositions may be used to treat a disease or disorder such as cancer, cystic fibrosis, or other genetic diseases.

BACKGROUND

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/337,196, filed on May 16, 2016, the entirety of whichis incorporated herein by reference.

1. Field

The present disclosure relates generally to the fields of lipids andnanoparticles. In particular, it relates to compositions which comprisesa nucleic acid. More particularly, it relates to lipid compositions forthe delivery of the nucleic acid.

2. Description of Related Art

Numerous genetic diseases can be corrected by nucleic acid therapeutics.However, these therapies require delivery systems to transport nucleicacid drugs into cells. There has been a continuous search for optimaldelivery carriers. Formulated lipid nanoparticles (LNPs) containing acationic/ionizable lipid, cholesterol, lipid PEG, and structural lipidssuch as DSPC are currently the most effective siRNA delivery system andare used in Phase 2 and 3 clinical trials. Yet, new lipids, dendrimers,and lipid-like materials are needed to address future therapeutictargets and overcome current limits with existing materials.

Materials that can deliver nucleic acids (siRNA, miRNA, mRNA, CRISPR,tRNA, sgRNA, tracRNA, etc.) are of therapeutic importance. Given thenumerous barriers to successful delivery, there remains a greattherapeutic need for new materials which can delivery nucleic acidtherapeutics.

SUMMARY

In some aspects, the present disclosure provides a compound of theformula:

wherein:

-   -   X₁ is —S(O)₂O⁻, —OP(O)OR_(e)O⁻, —(CHR_(f))₂C(O)O⁻, or        —NR_(g)R_(h)R_(i) ⁺, wherein:        -   R_(e), R_(g), R_(h), and R_(i) are each independently            hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        -   R_(f) is hydrogen, amino, hydroxy, or alkyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            acyl_((C≦12)), alkoxy_((C≦12)), acyloxy_((C≦12)),            amido_((C≦12)), alkoxy_((C≦12)), alkoxy_((C≦12)), or a            substituted version of any of the last ten groups; and        -   z is 1, 2, 3, or 4;    -   Y₁ is alkanediyl_((C≦12)), alkenediyl_((C≦12)),        arenediyl_((C≦12)), heteroarenediyl_((C≦12)),        heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),        -alkane-diyl_((C≦8))-heteroarenediyl_((C≦12)),        -alkanediyl_((C≦8))-heteroarenediyl_((C≦12))-alkanediyl_((C≦8)),        or a substituted version of any of these groups;    -   Z₁ is —N⁺R₃R₄— or —OP(O)O⁻O⁻    -   A is —NR_(a)—, —S—, or —O—; wherein:        -   R_(a) is hydrogen, alkyl_((C≦6)), or substituted            alkyl_((C≦6)), or R_(a) is taken together with either R₃ or            R₄ and is alkanediyl_((C≦8)), alkenediyl_((C≦8)),            alkoxydiyl_((C≦8)), alkylaminodiyl_((C≦8)), or a substituted            version of any of these groups;    -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))NR′R″, or a substituted version of any of            these groups wherein:        -   R′ and R″ are each independently hydrogen, alkyl_((C≦8)),            substituted alkyl_((C≦8)), or —Z₂A′R₇; wherein:            -   Z₂ is alkanediyl_((C≦6)), substituted                alkanediyl_((C≦6)), or a group of the formula:

-   -   -   -   wherein:                -   Z₅ and Z₆ are each independently alkanediyl_((C≦6))                    or substituted alkanediyl_((C≦6));                -   X₂ and X₃ are each independently —O—, —S—, or                    —NR_(m)—; wherein: R_(m) is hydrogen, alkyl_((C≦6)),                    or substituted alkyl_((C≦6)); and                -   a is 0, 1, 2, 3, 4, 5, or 6;            -   A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(j) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24));            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or

        -   R₅, R₆, and R₂ are each independently —Z₃A″R₈; wherein:            -   Z₃ is alkanediyl_((C≦6)), substituted                alkanediyl_((C≦6)), or a group of the formula:

-   -   -   -   wherein:                -   Z₇ and Z₈ are each independently alkanediyl_((C≦6))                    or substituted alkanediyl_((C≦6));                -   X₄ and X₅ are each independently —O—, —S—, or                    —NR_(n)—; wherein:                -    R_(n) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   b is 0, 1, 2, 3, 4, 5, or 6;            -   A″ is —CHR_(k)—, —S—, —C(O)O—, or —C(O)NR₁—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));

        -   q is 1, 2, or 3; and

        -   r is 1, 2, 3, or 4;

    -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a            substituted version of any of these groups;        -   R₉, R₁₀, and R₁₁ are each independently hydrogen,            alkyl_((C≦8)), substituted alkyl_((C≦8)), or —Z₄A′″R₁₂;            wherein:            -   Z₄ is alkanediyl_((C≦6)), substituted                alkanediyl_((C≦6)), or a group of the formula:

-   -   -   -   wherein:                -   Z₉ and Z₁₀ are each independently alkanediyl_((C≦6))                    or substituted alkanediyl_((C≦6));                -   X₆ and X₇ are each independently —O—, —S—, or —NR₀—;                    wherein:                -    R₀ is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   c is 0, 1, 2, 3, 4, 5, or 6;            -   A′″ is —CHR_(k)—, —S—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and

        -   x and y are 0, 1, 2, 3, or 4;

    -   R₃ and R₄ are each independently hydrogen, alkyl_((C≦6)), or        substituted alkyl_((C≦6)), or R₃ or R₄ are taken together with        R_(a) and is alkanediyl_((C≦8)), alkenediyl_((C≦8)),        alkoxydiyl_((C≦8)), alkylaminodiyl_((C≦8)), or a substituted        version of any of these groups; and

    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;

provided that if X₁ is positively charged then Z₁ is negatively charged,and if X₁ is negatively charged, then Z₁ is positively charged;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is further defined as:

wherein:

-   -   Y₁ is alkanediyl_((C≦12)), alkenediyl_((C≦12)),        arenediyl_((C≦12)), heteroarenediyl_((C≦12)),        heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))heterocycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),        -alkane-diyl_((C≦8))-heteroarenediyl_((C≦12)),        -alkanediyl_((C≦8))-heteroarenediyl_((C≦12))-alkanediyl_((C≦8)),        or a substituted version of any of these groups;    -   A is —NR_(a)—, —S—, or —O—; wherein:        -   R_(a) is hydrogen, alkyl_((C≦6)), or substituted            alkyl_((C≦6)), or R_(a) is taken together with either R₃ or            R₄ and is alkanediyl_((C≦8)), alkenediyl_((C≦8)),            alkoxydiyl_((C≦8)), alkylaminodiyl_((C≦8)), or a substituted            version of any of these groups;    -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((c≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))NR′R″, or a substituted version of any of            these groups wherein:        -   R′ and R″ are each independently hydrogen, alkyl_((C≦8)),            substituted alkyl_((C≦8)), —(CH₂)_(s)CH(OH)R₇,            —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NR_(b))R₇; wherein:            -   s is 1, 2, 3, or 4;            -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   R₅, R₆, and R₂ are each independently —(CH₂)_(t)CH(OH)R₈,            —(CH₂)_(t)C(O)OR₈, —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:            -   t is 1, 2, 3, or 4;            -   R_(c) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4;    -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), or a substituted version of any of            these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),            —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:            -   u is 1, 2, 3, or 4;            -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;    -   R₃ and R₄ are each independently hydrogen, alkyl_((C≦6)), or        substituted alkyl_((C≦6)), or R₃ or R₄ are taken together with        R_(a) and is alkanediyl_((C≦8)), alkenediyl_((C≦8)),        alkoxydiyl_((C≦8)), alkylaminodiyl_((C≦8)), or a substituted        version of any of these groups; and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments the compounds are further defined as:

wherein:

-   -   Y₁ is alkanediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),        or a substituted version of any of these groups;    -   A is —NR_(a)— or —O—; wherein:    -   R_(a) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)),        or R_(a) is taken together with either R₃ or R₄ and is        alkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),        alkylaminodiyl_((C≦8)), or a substituted version of any of these        groups;    -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))-NR′R″, or a substituted version of any            of these groups wherein:        -   R′ and R″ are each independently hydrogen, alkyl_((C≦8)),            substituted alkyl_((C≦8)), or —Z₂A′R₇; wherein:            -   Z₂ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(j) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24));            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   R₅, R₆, and R₂ are each independently —Z₃A″R₈; wherein:            -   Z₃ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4;    -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a            substituted version of any of these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or            —Z₄A′″R₁₂; wherein:            -   Z₄ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;    -   R₃ and R₄ are each independently hydrogen, alkyl_((C≦6)), or        substituted alkyl_((C≦6)), or R₃ or R₄ are taken together with        R_(a) and is alkanediyl_((C≦8)), alkenediyl_((C≦8)),        alkoxydiyl_((C≦8)), alkylaminodiyl_((C≦8)), or a substituted        version of any of these groups; and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the compounds are further defined as:

wherein:

-   -   Y₁ is alkanediyl_((C≦12)), heterocycloalkanediyl_((c≦12)),        -alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12)),        -alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),        or a substituted version of any of these groups;    -   A is —NR_(a)— or —O—; wherein:    -   R_(a) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)),        or R_(a) is taken together with either R₃ or R₄ and is        alkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),        alkylaminodiyl_((C≦8)), or a substituted version of any of these        groups;    -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))-NR′R″, or a substituted version of any            of these groups wherein:            -   R′ and R″ are each independently hydrogen,                alkyl_((C≦8)), substituted alkyl_((C≦8)),                —(CH₂)_(s)CH(OH)R₇, —(CH₂)_(s)C(O)OR₇, or                —(CH₂)_(s)C(O)(NR_(b))R₇; wherein:                -   s is 1, 2, 3, or 4;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                    alkenyl_((C6-24)), substituted alkenyl_((C6-24)), or        -   R₅, R₆, and R₂ are each independently —(CH₂)_(t)CH(OH)R₈,            —(CH₂)_(t)C(O)OR₈, —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:            -   t is 1, 2, 3, or 4;            -   R_(c) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl₍₆₋₂₄₎,                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4;    -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), or a substituted version of any of            these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),            —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:            -   u is 1, 2, 3, or 4;            -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;    -   R₃ and R₄ are each independently hydrogen, alkyl_((C≦6)), or        substituted alkyl_((C≦6)), or R₃ or R₄ are taken together with        R_(a) and is alkanediyl_((C≦8)), alkenediyl_((C≦8)),        alkoxydiyl_((C≦8)), alkylaminodiyl_((C≦8)), or a substituted        version of any of these groups; and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))-NR′R″, or a substituted version of any            of these groups wherein:        -   R′ and R″ are each independently hydrogen, alkyl_((C≦8)),            substituted alkyl_((C≦8)), or —Z₂A′R₇; wherein:            -   Z₂ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(j) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24));            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   R₅, R₆, and R₂ are each independently —Z₃A″R₈; wherein:            -   Z₃ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A″ is —-CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4;    -   R_(a), R₃, and R₄ are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)); and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))-NR′R″, or a substituted version of any            of these groups wherein:            -   R′ and R″ are each independently hydrogen,                alkyl_((C≦8)), substituted alkyl_((C≦8)),                —(CH₂)_(s)CH(OH)R₇, —(CH₂)_(s)C(O)OR₇, or                —(CH₂)_(s)C(O)(NR_(b))R₇; wherein:                -   s is 1, 2, 3, or 4;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                    alkenyl_((C6-24)), substituted alkenyl_((C6-24)), or        -   R₅, R₆, and R₂ are each independently —(CH₂)_(t)CH(OH)R₈,            —(CH₂)_(t)C(O)OR₈, —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:            -   t is 1, 2, 3, or 4;            -   R_(c) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4;    -   R_(a), R₃, and R₄ are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)); and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In other        embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))-NR′R″, or a substituted version of any            of these groups wherein:            -   R′ and R″ are each independently hydrogen,                alkyl_((C≦8)), substituted alkyl_((C≦8)),                —(CH₂)_(s)CH(OH)R₇, —(CH₂)_(s)C(O)OR₇, or                —(CH₂)_(s)C(O)(NR_(b))R₇; wherein:                -   s is 1, 2, 3, or 4;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                    alkenyl_((C6-24)), substituted alkenyl_((C6-24)), or        -   R₅, R₆, and R₂ are each independently —(CH₂)_(t)CH(OH)R₈,            —(CH₂)_(t)C(O)OR₈, —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:            -   t is 1, 2, 3, or 4;            -   R_(c) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4; and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅, R₆, and R₂ are each independently hydrogen or            alkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,            -alkanediyl_((C≦6))-alkylamino_((C≦8)),            -alkanediyl_((C≦6))-dialkylamino_((C≦12)),            -alkanediyl_((C≦6))-NR′R″, or a substituted version of any            of these groups wherein:            -   R′ and R″ are each independently are each independently                hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),                —(CH₂)_(s)CH(OH)R₇, —(CH₂)_(s)C(O)OR₇, or                —(CH₂)_(s)C(O)(NR_(b))R₇; wherein:            -   s is 1, 2, 3, or 4;            -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   R₅, R₆, and R₂ are each independently —(CH₂)_(t)CH(OH)R₈,            —(CH₂)_(t)C(O)OR₈, —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:            -   t is 1, 2, 3, or 4;            -   R_(c) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   q is 1, 2, or 3; and        -   r is 1, 2, 3, or 4;            or a pharmaceutically acceptable salt thereof. In some            embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅ is —(CH₂)_(t)CH(OH)R₈, —(CH₂)_(t)C(O)OR₈,            —(CH₂)_(t)C(O)(NH)R₈; wherein:            -   t is 1 or 2; and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));        -   R₆ is alkyl_((C≦8)) or substituted alkyl_((C≦8)); and        -   R₂ is -alkanediyl_((C≦6))-NR′R″ or a substituted version of            this group wherein:            -   R′ and R″ are each independently —(CH₂)_(s)CH(OH)R₇,                —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NH)R₇; wherein:            -   s is 1 or 2; and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or        -   q is 1 or 2; and        -   r is 1 or 2;            or a pharmaceutically acceptable salt thereof. In some            embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅ is alkyl_((C≦8)) or substituted alkyl_((C≦8));        -   R₆ is -alkanediyl_((C≦6))-NR′R″ or a substituted version of            this group wherein:            -   R′ and R″ are each independently alkyl_((C≦8)),                substituted alkyl_((C≦8)), —(CH₂)_(s)CH(OH)R₇,                —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NH)R₇; wherein:            -   s is 1 or 2; and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   R₂ is -alkanediyl_((C≦6))-NR′R″ or a substituted version of            this group wherein:            -   R′ and R″ are each independently alkyl_((C≦8)),                substituted alkyl_((C≦8)), —(CH₂)_(s)CH(OH)R₇,                —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NH)R₇; wherein:            -   s is 1 or 2; and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));        -   q is 1 or 2; and        -   r is 1 or 2;            or a pharmaceutically acceptable salt thereof. In some            embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   R₅ is —(CH₂)_(t)CH(OH)R₈, —(CH₂)_(t)C(O)OR₈,            —(CH₂)_(t)C(O)(NH)R₈; wherein:            -   t is 1 or 2; and            -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                -alkenyl_((C6-24)), substituted alkenyl_((C6-24));        -   R₆ is -alkanediyl_((C≦6))-NR′R″ or a substituted version of            this group; wherein:            -   R′ and R″ are each independently —(CH₂)_(s)CH(OH)R₇,                —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NH)R₇; wherein:            -   s is 1 or 2; and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   R₂ is -alkanediyl_((C≦6))-NR′R″ or a substituted version of            this group; wherein:            -   R′ and R″ are each independently —(CH₂)_(s)CH(OH)R₇,                —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NH)R₇; wherein:            -   s is 1 or 2; and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));        -   q is 1 or 2; and        -   r is 1 or 2;            or a pharmaceutically acceptable salt thereof. In some            embodiments the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a            substituted version of any of these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or            —Z₄A′″R₁₂; wherein:            -   Z₄ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C≦24)); and        -   x and y are 1, 2, 3, or 4;    -   R_(a), R₃, and R₄ are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)); and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof.        In other embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), or a substituted version of any of            these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),            —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:            -   u is 1, 2, 3, or 4;            -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;    -   R_(a), R₃, and R₄ are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)); and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), or a substituted version of any of            these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),            —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:            -   u is 1, 2, 3, or 4;            -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4; and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), or a substituted version of any of            these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),            —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:            -   u is 1, 2, 3, or 4;            -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;            or a pharmaceutically acceptable salt thereof. In some            embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is heterocycloalkanediyl_((C≦12)) or substituted            heterocycloalkanediyl_((C≦12));        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),            —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:            -   u is 1, 2, 3, or 4;            -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;            or a pharmaceutically acceptable salt thereof. In some            embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is heterocycloalkanediyl_((C≦12)) or substituted            heterocycloalkanediyl_((C≦12));        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,            —(CH₂)_(u)C(O)(NH)R₁₂; wherein:            -   u is 1 or 2; and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;            or a pharmaceutically acceptable salt thereof. In some            embodiments the compounds are further defined as:

wherein:

-   -   R_(f) is hydrogen, amino, hydroxy, or alkyl_((C≦12)),        aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl(_(c<)12),        acyl_((C≦12)), alkoxy_((C≦12)), acyloxy_((C≦12)),        amido_((C≦12)), alkoxy_((C≦12)), alkoxy_((C≦12)), or a        substituted version of any of the last ten groups;    -   z is 1, 2, 3, or 4;    -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a            substituted version of any of these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or            —Z₄A′|R₁₂; wherein:            -   Z₄ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;    -   R_(a), R₃, and R₄ are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)); and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments the compounds are further defined as:

wherein:

-   -   R_(e) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));    -   R₁ is a group of the formula:

-   -   wherein:        -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),            heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a            substituted version of any of these groups;        -   R₉, R₁₀, and R₁₁ are each independently selected from            hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or            —Z₄A′″R₁₂; wherein:            -   Z₄ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;                -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24)); and            -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and        -   x and y are 1, 2, 3, or 4;    -   R_(a), R₃, and R₄ are each independently hydrogen,        alkyl_((C≦6)), or substituted alkyl_((C≦6)); and    -   m, n, and p are each independently an integer selected from 0,        1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof.

In some embodiments, R_(a) is hydrogen. In other embodiments, R_(a) isalkyl_((C≦8)) or substituted alkyl_((C≦8)). In some embodiments, R₃ ishydrogen. In other embodiments, R₃ is alkyl_((C≦8)) or substitutedalkyl_((C≦8)). R₃ may be alkyl_((C≦8)) such as methyl. In someembodiments, R₄ is hydrogen. In other embodiments, R₄ is alkyl_((C≦8))or substituted alkyl_((C≦8)). R₄ may be alkyl_((C≦8)) such as methyl.

In some embodiments, m is 1 or 2. In one instance, m is 1. In anotherinstance, m is 2. In some embodiments, n is 2 or 3. In one instance, nis 2. In another instance, n is 3. In some embodiments, p is 1, 2, or 3.In one instance, p is 1. In another instance, p is 2. In yet anotherinstance, p is 3.

In some embodiments, R₁ is a group of the formula:

wherein:

-   -   R₅, R₆, and R₂ are each independently hydrogen or alkyl_((C≦8)),        -alkanediyl_((c≦6))-NH₂, -alkanediyl_((C≦6))alkylamino_((C≦8)),        -alkanediyl_((C≦6))-dialkylamino_((C≦12)),        -alkanediyl_((C≦6))-NR′R″, or a substituted version of any of        these groups wherein:        -   R′ and R″ are each independently hydrogen, alkyl_((C≦8)),            substituted alkyl_((C≦8)), —Z₂A′R₇;            -   wherein:            -   Z₂ is alkanediyl_((C≦4)) or substituted                alkanediyl_((C≦4));            -   A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;                -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                    alkyl_((C≦6)); and                -   R_(j) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),                    or substituted acyloxy_((C≦24));            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));            -   or    -   R₅, R₆, and X₁ are each independently —Z₃A″R₈; wherein:        -   Z₃ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));        -   A″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;            -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or                substituted acyloxy_((C≦24)); and        -   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),            alkenyl_((C6-24)), substituted alkenyl_((C6-24));    -   q is 1, 2, or 3; and    -   r is 1, 2, 3, or 4.

In some embodiments, R₁ is a group of the formula:

wherein:

-   -   R₅, R₆, and R₂ are each independently hydrogen or alkyl_((C≦8)),        -alkanediyl_((C≦6))-NH₂, -alkanediyl_((C≦6))-alkylamino_((C≦8)),        -alkanediyl_((C≦6))-dialkylamino_((C≦12)),        -alkanediyl_((C≦6))-NR′R″, or a substituted version of any of        these groups wherein:        -   R′ and R″ are each independently hydrogen, alkyl_((C≦8)),            substituted alkyl_((C≦8)), —(CH₂)_(s)CH(OH)R₇,            —(CH₂)_(s)C(O)OR₇, or —(CH₂)_(s)C(O)(NR_(b))R₇; wherein:            -   s is 1, 2, 3, or 4;            -   R_(b) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),                alkenyl_((C6-24)), substituted alkenyl_((C6-24));            -   or    -   R₅, R₆, and X₁ are each independently —(CH₂)_(t)CH(OH)R₈,        —(CH2)_(t)C(O)OR₈, —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:        -   t is 1, 2, 3, or 4;        -   R_(c) is hydrogen, alkyl_((C≦6)), or substituted            alkyl_((C≦6)); and        -   R_(s) is alkyl_((C6-24)), substituted alkyl_((C6-24)),            alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or    -   q is 1, 2, or 3; and    -   r is 1, 2, 3, or 4.

In some embodiments, q is 1 or 2. In one instance, q is 1. In anotherinstance, q is 2. In some embodiments, r is 1 or 2. In one instance, ris 1. In another instance, r is 2. In some embodiments, R₅ is hydrogen.In other embodiments, R₅ is alkyl_((C≦8)) or substituted alkyl_((C≦8)).R₅ may be alkyl_((C≦8)) such as methyl or isopropyl.

In some embodiments, R₅ is further defined as —Z₃A″R₈ wherein:

-   Z₃ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));-   A″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;    -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        and    -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or        substituted acyloxy_((C≦24)); and-   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),    alkenyl_((C6-24)), substituted alkenyl_((C6-24)).

In some embodiments, Z₃ is alkanediyl_((C1-2)). In one instance, Z₃ is—CH₂—. In some embodiments, Z₃ is substituted alkanediyl_((C1-2)). Inone instance, Z₃ is —CH₂CH(OH). In some embodiments, A″ is —CHR_(k)—. Inone instance, R_(k) is hydroxy. In some embodiments, R_(k) isacyloxy_((C≦24)) or substituted acyloxy_((C≦24)). In some embodiments,R_(k) is acyloxy_((C1-8)) or substituted acyloxy_((C1-8)). In someembodiments, R_(k) is acyloxy_((C≦12-24)) or substitutedacyloxy_((C≦12-24)). In one instance, A″ is —C(O)O—. In anotherinstance, A″ is —C(O)NH—.

In other embodiments, R₅ is —(CH₂)_(t)CH(OH)R₈, —(CH₂)_(t)C(O)OR₈, or—(CH₂)_(t)C(O)(NR_(c))R₈; wherein:

t is 1, 2, 3, or 4;

R_(c) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and

R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)).

In some embodiments, R₅ is —(CH₂)_(t)CH(OH)R₈. In other embodiments, R₅is —(CH₂)_(t)C(O)OR₈. In other embodiments, R₅ is—(CH₂)_(t)C(O)(NR_(c))R₈. In some embodiments, t is 1 or 2. In oneinstance, t is 1. In another instance, t is 2. In some embodiments,R_(c) is hydrogen. In other embodiments, R_(c) is alkyl_((C≦6)) orsubstituted alkyl_((C≦6)). In some embodiments, R₈ is alkyl_((C6-24)) orsubstituted alkyl_((C6-24)). R₈ may be alkyl_((C6-24)) such as octyl,decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₈ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₆ is —Z₃A″R₈; wherein:

-   Z₃ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));-   A″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;    -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        and    -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or        substituted acyloxy_((C≦24)); and-   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),    alkenyl_((C6-24)), substituted alkenyl_((C6-24)).

In some embodiments, Z₃ is alkanediyl_((C1-2)). In one instance, Z₃ is—CH₂—. In some embodiments, Z₃ is substituted alkanediyl_((C1-2)). Inone instance, Z₃ is —CH₂CH(OH). In some embodiments, A″ is —CHR_(k)—. Inone instance, R^(k) is hydroxy. In some embodiments, R_(k) isacyloxy_((C≦24)) or substituted acyloxy_((C≦24)). In some embodiments,R_(k) is acyloxy_((C1-8)) or substituted acyloxy_((C1-8)). In someembodiments, R_(k) is acyloxy_((C≦12-24)) or substitutedacyloxy_((C≦12-24)). In one instance, A″ is —C(O)O—. In anotherinstance, A″ is —C(O)NH—. In some embodiments, R₈ is alkyl_((C6-24)) orsubstituted alkyl_((C6-24)). R₈ may be alkyl_((C6-24)) such as octyl,decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₈ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₆ is hydrogen. In other embodiments, R₆ isalkyl_((C≦8)) or substituted alkyl_((C≦8)). R₆ may be alkyl_((C≦8)) suchas methyl or isopropyl. In other embodiments, R₆ is —(CH₂)_(t)CH(OH)R₈,—(CH₂)_(t)C(O)OR₈, or —(CH₂)_(t)C(O)(NR_(c))R₈; wherein:

t is 1, 2, 3, or 4;

R_(c) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and

R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)).

In some embodiments, R₆ is —(CH₂)_(t)CH(OH)R₈. In other embodiments, R₆is —(CH₂)_(t)C(O)OR₈. In other embodiments, R₆ is—(CH₂)_(t)C(O)(NR_(c))R₈. In some embodiments, t is 1 or 2. In oneinstance, t is 1. In another instance, t is 2. In some embodiments,R_(c) is hydrogen. In some embodiments, R_(c) is alkyl_((C≦6)) orsubstituted alkyl_((C≦6)). In some embodiments, R₈ is alkyl_((C6-24)) orsubstituted alkyl_((C6-24)). R₈ may be alkyl_((C6-24)) such as octyl,decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₈ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₆ is -alkanediyl_((C≦6))-NH₂,-alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))dialkylamino_((C≦12)), or a substituted version ofany of these groups. In some embodiments, R₆ is -alkanediyl_((C≦6))-NH₂or a substituted version of this group such as —CH₂CH₂NH₂. In otherembodiments, R₆ is -alkanediyl_((C≦6))-alkylamino_((C≦8)) or asubstituted version of this group such as —CH₂CH₂NHMe or —CH₂CH₂NHiPr.In other embodiments, R₆ is -alkanediyl_((C≦6))-dialkylamino_((C≦8)) ora substituted version of this group.

In some embodiments, R₂ is hydrogen. In other embodiments, R₂ isalkyl_((C≦8)) or substituted alkyl_((C≦8)). R₂ may be alkyl_((C≦8)) suchas methyl or isopropyl.

In some embodiments, R₂ is —Z₃A″R₈; wherein:

-   Z₃ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));-   A″ is —CHR_(k)—, —C(O)O—, or —C(O)NR₁—;    -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        and    -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or        substituted acyloxy_((C≦24)); and-   R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)),    alkenyl_((C6-24)), substituted alkenyl_((C6-24)).

In some embodiments, Z₃ is alkanediyl_((C1-2)). In one instance, Z₃ is—CH₂—. In some embodiments, Z₃ is substituted alkanediyl_((C1-2)). Inone instance, Z₃ is —CH₂CH(OH). In some embodiments, A″ is —CHR_(k)—. Inone instance, R_(k) is hydroxy. In some embodiments, R_(k) isacyloxy_((C≦24)) or substituted acyloxy_((C≦24)). In some embodiments,R_(k) is acyloxy_((C1-8)) or substituted acyloxy_((C1-8)). In someembodiments, R_(k) is acyloxy_((C≦12-24)) or substitutedacyloxy_((C≦12-24)). In one instance, A″ is —C(O)O—. In anotherinstance, A″ is —C(O)NH—. In some embodiments, R₈ is alkyl_((C6-24)) orsubstituted alkyl_((C6-24)). R₈ may be alkyl_((C6-24)) such as octyl,decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₈ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₂ is -alkanediyl_((C≦6))-NH₂,-alkanediyl_((C≦6))alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), or a substituted version ofany of these groups. In some embodiments, R₂ is -alkanediyl_((C≦6))-NH₂or a substituted version of this group such as —CH₂CH₂NH₂. In otherembodiments, R₂ is -alkanediyl_((C≦6))alkylamino_((C≦8)) or asubstituted version of this group such as —CH₂CH₂NHMe or —CH₂CH₂NHiPr.In other embodiments, R₂ is alkanediyl_((C≦6))dialkylamino_((C≦8)) or asubstituted version of this group.

In other embodiments, R₂ is —(CH₂)_(t)CH(OH)R₈, —(CH₂)_(t)C(O)OR₈, or—(CH₂)_(t)C(O)(NR_(c))R₈; wherein:

t is 1, 2, 3, or 4;

R_(c) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and

R₈ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)).

In other embodiments, R₂ is —(CH₂)_(t)CH(OH)R₈. In other embodiments, R₂is —(CH₂)_(t)C(O)OR₈. In other embodiments, R₂ is—(CH₂)_(t)C(O)(NR_(c))R₈. In some embodiments, t is 1 or 2. In oneinstance, t is 1. In another instance, t is 2. In some embodiments,R_(c) is hydrogen. In some embodiments, R_(c) is alkyl_((C≦6)) orsubstituted alkyl_((C≦6)). In some embodiments, R₈ is alkyl_((C6-24)) orsubstituted alkyl_((C6-24)). R₈ may be alkyl_((C6-24)) such as octyl,decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₈ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In other embodiments, R₂ is -alkanediyl_((C≦6))-NH₂,alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), or a substituted version ofany of these groups. In some embodiments, R₂ is -alkanediyl_((C≦6))-NH₂or a substituted version of this group such as —CH₂CH₂NH₂. In otherembodiments, R₂ is -alkanediyl_((C≦6))-alkylamino_((C≦8)) or asubstituted version of this group such as —CH₂CH₂NHMe or —CH₂CH₂NHiPr.In other embodiments, R₂ is -alkanediyl_((C≦6))dialkylamino_((C≦8)) or asubstituted version of this group.

In some embodiments, R₁ is a group of the formula:

wherein:

-   -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),        heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a substituted        version of any of these groups;    -   R₉, R₁₀, and R₁₁ are each independently selected from hydrogen,        alkyl_((C≦8)), substituted alkyl_((C≦8)), or —Z₄A′″R₁₂; wherein:        -   Z₄ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));        -   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR₁—;            -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted                alkyl_((C≦6)); and            -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or                substituted acyloxy_((C≦24)); and        -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),            alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and    -   x and y are 1, 2, 3, or 4.

In some embodiments, R₁ is a group of the formula:

wherein:

-   -   Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),        heteroarenediyl_((C≦12)), or a substituted version of any of        these groups;    -   R₉, R₁₀, and R₁₁ are each independently selected from hydrogen,        alkyl_((C≦8)), substituted alkyl_((C≦8)), —(CH₂)_(u)—CH(OH)R₁₂,        —(CH₂)_(u)C(O)OR₁₂, —(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:        -   u is 1, 2, 3, or 4;        -   R_(d) is hydrogen, alkyl_((C≦6)), or substituted            alkyl_((C≦6)); and        -   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),            alkenyl_((C6-24)), substituted alkenyl_((C6-24)); and    -   x and y are 1, 2, 3, or 4.

In some embodiments, Y₂ is heterocycloalkanediyl_((C≦12)) or substitutedheterocycloalkanediyl_((C≦12)). Y₂ may be heterocycloalkanediyl_((C≦12))such as piperazindiyl. In other embodiments, Y₂ isheteroarenediyl_((C≦12)) or substituted heteroarenediyl_((C≦12)). Inother embodiments, Y₂ is arenediyl_((C≦12)) or substitutedarenediyl_((C≦12)). In some embodiments, Y₂ is alkoxydiyl_((C≦12)) orsubstituted alkoxyldiyl_((C≦12)). In some embodiments, x is 2 or 3. Inone instance, x is 2. In another instance, x is 3. In some embodiments,y is 2 or 3. In one instance, y is 2. In another instance, y is 3.

In some embodiments, R₉ is —Z₄A′″R₁₂; wherein:

-   Z₄ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));-   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;    -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        and    -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or        substituted acyloxy_((C≦24)); and-   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),    alkenyl_((C6-24)), substituted alkenyl_((C6-24)).

In some embodiments, Z₄ is alkanediyl_((C1-2)). In one instance, Z₄ is—CH₂—. In some embodiments, Z₄ is substituted alkanediyl_((C1-2)). Inone instance, Z₄ is —CH₂CH(OH). In some embodiments, A′″ is —CHR_(k)—.In one instance, R_(k) is hydroxy. In some embodiments, R_(k) isacyloxy_((C≦24)) or substituted acyloxy_((C≦24)). In some embodiments,R_(k) is acyloxy_((C1-8)) or substituted acyloxy_((C1-8)). In someembodiments, R_(k) is acyloxy_((C≦12-24)) or substitutedacyloxy_((C≦12-24)). In one instance, A′″ is —C(O)O—. In anotherinstance, A′″ is —C(O)NH—. In some embodiments, R₁₂ is alkyl_((C6-24))or substituted alkyl_((C6-24)). R₁₂ may be alkyl_((C6-24)) such asoctyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₁₂ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₉ is —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,—(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:

u is 1, 2, 3, or 4;

R_(d) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and

R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)).

In some embodiments, R₉ is —(CH₂)_(u)CH(OH)R₁₂. In other embodiments, R₉is —(CH₂)_(u)C(O)OR₁₂. In other embodiments, R₉ is—(CH₂)_(u)C(O)(NR_(d))R₁₂. In some embodiments, u is 1, 2, or 3. In someembodiments, u is 1 or 2. In one instance, u is 1. In another instance,u is 2. In some embodiments, R_(d) is hydrogen. In other embodiments,R_(d) is alkyl_((C≦6)) or substituted alkyl_((C≦6)). In someembodiments, R₁₂ is alkyl_((C6-24)) or substituted alkyl_((C6-24)). R₁₂may be alkyl_((C6-24)) such as octyl, decyl, dodecyl, tetradecyl,hexadecyl, or octadecyl. In other embodiments, R₁₂ is alkenyl_((C6-24))or substituted alkenyl_((C6-24)).

In some embodiments, R₁₀ is —Z₄A′″R₁₂; wherein:

-   Z₄ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));-   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;    -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        and    -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or        substituted acyloxy_((C≦24)); and-   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),    alkenyl_((C6-24)), substituted alkenyl_((C6-24)).

In some embodiments, Z₄ is alkanediyl_((C1-2)). In one instance, Z₄ is—CH₂—. In some embodiments, Z₄ is substituted alkanediyl_((C1-2)). Inone instance, Z₄ is —CH₂CH(OH). In some embodiments, A′″ is —CHR_(k)—.In one instance, R_(k) is hydroxy. In some embodiments, R_(k) isacyloxy_((C≦24)) or substituted acyloxy_((C≦24)). In some embodiments,R_(k) is acyloxy_((C1-8)) or substituted acyloxy_((C1-8)). In someembodiments, R_(k) is acyloxy_((C≦12-24)) or substitutedacyloxy_((C≦12-24)). In one instance, A′″ is —C(O)O—. In anotherinstance, A′″ is —C(O)NH—. In some embodiments, R₁₂ is alkyl_((C6-24))or substituted alkyl_((C6-24)). R₁₂ may be alkyl_((C6-24)) such asoctyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₁₂ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₁₀ is —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,—(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:

u is 1, 2, 3, or 4;

R_(d) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and

R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)).

In some embodiments, R₁₀ is —(CH₂)_(u)CH(OH)R₁₂. In other embodiments,R₁₀ is —(CH₂)_(u)C(O)OR₁₂. In other embodiments, R₁₀ is—(CH₂)_(u)C(O)(NR_(d))R₁₂. In some embodiments, u is 1, 2, or 3. In someembodiments, u is 1 or 2. In one instance, u is 1. In another instance,u is 2. In some embodiments, R_(d) is hydrogen. In other embodiments,R_(d) is alkyl_((C≦6)) or substituted alkyl_((C≦6)). In someembodiments, R₁₂ is alkyl_((C6-24)) or substituted alkyl_((C6-24)). R₁₂may be alkyl_((C6-24)) such as octyl, decyl, dodecyl, tetradecyl,hexadecyl, or octadecyl. In other embodiments, R₁₂ is alkenyl_((C6-24))or substituted alkenyl_((C6-24)).

In some embodiments, R₁₁ is —Z₄A′″R₁₂; wherein:

-   Z₄ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4));-   A′″ is —CHR_(k)—, —C(O)O—, or —C(O)NR₁—;    -   R_(l) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));        and    -   R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)), or        substituted acyloxy_((C≦24)); and-   R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)),    alkenyl_((C6-24)), substituted alkenyl_((C6-24)).

In some embodiments, Z₄ is alkanediyl_((C1-2)). In one instance, Z₄ is—CH₂—. In some embodiments, Z₄ is substituted alkanediyl_((C1-2)). Inone instance, Z₄ is —CH₂CH(OH). In some embodiments, A′″ is —CHR_(k)—.In one instance, R_(k) is hydroxy. In some embodiments, R_(k) isacyloxy_((C≦24)) or substituted acyloxy_((C≦24)). In some embodiments,R_(k) is acyloxy_((C1-8)) or substituted acyloxy_((C1-8)). In someembodiments, R_(k) is acyloxy_((C≦12-24)) or substitutedacyloxy_((C≦12-24)). In one instance, A′″ is —C(O)O—. In anotherinstance, A′″ is —C(O)NH—. In some embodiments, R₁₂ is alkyl_((C6-24))or substituted alkyl_((C6-24)). R₁₂ may be alkyl_((C6-24)) such asoctyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl. In someembodiments, R₁₂ is alkenyl_((C6-24)) or substituted alkenyl_((C6-24)).

In some embodiments, R₁₁ is —(CH₂)_(u)CH(OH)R₁₂, —(CH₂)_(u)C(O)OR₁₂,—(CH₂)_(u)C(O)(NR_(d))R₁₂; wherein:

u is 1, 2, 3, or 4;

R_(d) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and

R₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)).

In some embodiments, R₁₁ is —(CH₂)_(u)CH(OH)R₁₂. In other embodiments,R₁₁ is —(CH₂)_(u)C(O)OR₁₂. In other embodiments, R₁₁ is—(CH₂)_(u)C(O)(NR_(d))R₁₂. In some embodiments, u is 1, 2, or 3. In someembodiments, u is 1 or 2. In one instance, u is 1. In another instance,u is 2. In some embodiments, R_(d) is hydrogen. In other embodiments,R_(d) is alkyl_((C≦6)) or substituted alkyl_((C≦6)). In someembodiments, R₁₂ is alkyl_((C6-24)) or substituted alkyl_((C6-24)). R₁₂may be alkyl_((C6-24)) such as octyl, decyl, dodecyl, tetradecyl,hexadecyl, or octadecyl. In other embodiments, R₁₂ is alkenyl_((C6-24))or substituted alkenyl_((C6-24)).

In yet another aspect, the present disclosure provides compounds of theformula:

wherein:

-   -   R₁, R₂, and R₃ are each independently hydrogen, alkyl_((C≦6)),        substituted alkyl_((C≦6)), or a group of the formula:

-   -   wherein:        -   R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),            substituted alkyl_((C≦6)), or a group of the formula:

-   -   -   wherein:            -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),                acyloxy_((C≦8)), or a substituted version of either of                these groups; and            -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a                substituted version of either group;        -   q is 1, 2, or 3; and        -   r is 0, 1, 2, 3, or 4;

    -   R₄, R₅, and R₆ are each independently hydrogen, alkyl_((C≦6)),        or substituted alkyl_((C≦6)), or R₄ is taken together with        either R₅ or R₆ and is alkanediyl_((C≦12)), alkoxydiyl_((C≦12)),        alkylaminodiyl_((C≦12)), or a substituted version of any of        these groups; and

    -   m and n are each independently 1, 2, 3, 4, or 5;        or a pharmaceutically acceptable salt thereof In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁ is hydrogen, alkyl_((C≦6)), substituted alkyl_((C≦6)), or a        group of the formula:

-   -   -   wherein:        -   R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),            substituted alkyl_((C≦6)), or a group of the formula:

-   -   -   wherein:            -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),                acyloxy_((C≦8)), or a substituted version of either of                these groups; and            -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a                substituted version of either group;        -   q is 1, 2, or 3; and        -   r is 0, 1, 2, 3, or 4;

    -   R₄, R₅, and R₆ are each independently hydrogen, alkyl_((C≦6)),        or substituted alkyl_((C≦6)), or R₄ is taken together with        either R₅ or R₆ and is alkanediyl_((C≦12)), alkoxydiyl_((C≦12)),        alkylaminodiyl_((C≦12)), or a substituted version of any of        these groups; and

    -   m and n are each independently 1, 2, 3, 4, or 5;        or a pharmaceutically acceptable salt thereof In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁, R₂, and R₃ are each independently hydrogen, alkyl_((C≦6)),        substituted alkyl_((C≦6)), or a group of the formula:

-   -   wherein:        -   R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),            substituted alkyl_((C≦6)), or a group of the formula:

-   -   -   wherein:            -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),                acyloxy_((C≦8)), or a substituted version of either of                these groups; and            -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a                substituted version of either group;        -   q is 1, 2, or 3; and        -   r is 0, 1, 2, 3, or 4;

    -   R₅ and R₆ are each independently hydrogen, alkyl_((C≦6)), or        substituted alkyl_((C≦6)); and

    -   m and n are each independently 1, 2, 3, 4, or 5;        or a pharmaceutically acceptable salt thereof In some        embodiments, the compounds are further defined as:

wherein:

-   -   R₁, R₂, and R₃ are each independently hydrogen, alkyl_((C≦6)),        substituted alkyl_((C≦6)), or a group of the formula:

-   -   -   wherein:        -   R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),            substituted alkyl_((C≦6)), or a group of the formula:

-   -   -   wherein:            -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),                acyloxy_((C≦8)), or a substituted version of either of                these groups; and            -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a                substituted version of either group;        -   q is 1, 2, or 3; and        -   r is 0, 1, 2, 3, or 4;

    -   R₅ and R₆ are each independently hydrogen, alkyl_((C≦6)), or        substituted alkyl_((C≦6)); and

    -   m is 1, 2, 3, 4, or 5;        or a pharmaceutically acceptable salt thereof.

In some embodiments, R₂ is hydrogen. In other embodiments, R₂ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₂ may be alkyl_((C≦6)) suchas methyl or ethyl. In other embodiments, R₂ is a group of the formula:

wherein:

-   -   R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),        substituted alkyl_((C≦6)), or a group of the formula:

-   -   wherein:        -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),            acyloxy_((C≦8)), or a substituted version of either of these            groups; and        -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted            version of either group;    -   q is 1, 2, or 3; and    -   r is 0, 1, 2, 3, or 4.

In some embodiments, R₇ is hydrogen. In other embodiments, R₇ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₇ may be alkyl_((C≦6)) suchas methyl or ethyl. In other embodiments, R₇ is

wherein:

-   -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),        acyloxy_((C≦8)), or a substituted version of either of these        groups; and    -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted        version of either group.

In some embodiments, R₉ is halo such as chloro or bromo. In otherembodiments, R₉ is hydroxy. In other embodiments, R₉ is alkoxy_((C≦8))or substituted alkoxy_((C≦8)). R₉ may be alkoxy_((C≦8)) such as methoxy.In some embodiments, R₉ is acyloxy_((C≦8)) or substitutedacyloxy_((C≦8)). R₉ may be acyloxy_((C≦8)) such as acetoxy orpivaloyloxy. In some embodiments, R₁₀ is alkyl_((C≦24)) or substitutedalkyl_((C≦24)) such as octyl, decyl, dodecyl, tetradecyl, hexadecyl, oroctadecyl. In other embodiments, R₁₀ is alkenyl_((C≦24)) or substitutedalkenyl_((C≦24)).

In some embodiments, R₈ is hydrogen. In other embodiments, R₈ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₈ may be alkyl_((C≦6)) suchas methyl or ethyl. In some embodiments, R₈ is

wherein:

-   -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),        acyloxy_((C≦8)), or a substituted version of either of these        groups; and    -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted        version of either group.

In some embodiments, R₉ is halo such as chloro or bromo. In otherembodiments, R₉ is hydroxy. In other embodiments, R₉ is alkoxy_((C≦8))or substituted alkoxy_((C≦8)). R₉ may be alkoxy_((C≦8)) such as methoxy.In some embodiments, R₉ is acyloxy_((C≦8)) or substitutedacyloxy_((C≦8)). R₉ may be acyloxy_((C≦8)) such as acetoxy orpivaloyloxy. In some embodiments, R₁₀ is alkyl_((C≦24)) or substitutedalkyl_((C≦24)) such as octyl, decyl, dodecyl, tetradecyl, hexadecyl, oroctadecyl. In other embodiments, Rio is alkenyl_((C≦24)) or substitutedalkenyl_((C≦24)).

In some embodiments, q is 1 or 2. In one instance, q is 1. In anotherinstance, q is 2. In some embodiments, r is 1, 2, or 3. In one instance,r is 1. In another instance, r is 2. In another instance, r is 3.

In some embodiments, R₃ is hydrogen. In other embodiments, R₃ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₃ may be alkyl_((C≦6)) suchas methyl or ethyl. In some embodiments, R₄ is hydrogen. In otherembodiments, R₄ is alkyl_((C≦6)) or substituted alkyl_((C≦6)). In someembodiments, R₅ is hydrogen. In other embodiments, R₅ is alkyl_((C≦6))or substituted alkyl_((C≦6)). R₅ may be alkyl_((C≦6)) such as methyl orethyl. In some embodiments, R₆ is hydrogen. In other embodiments, R₆ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₆ may be alkyl_((C≦6)) suchas methyl or ethyl. In some embodiments, m is 2, 3, or 4. In oneinstance, m is 2. In another instance, m is 3. In another instance, m is4. In some embodiments, n is 2, 3, or 4. In one instance, n is 2. Inanother instance, n is 3. In yet another instance, n is 4.

In some embodiments, R₁ is hydrogen. In other embodiments, R₁ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₁ may be alkyl_((C≦6)) suchas methyl or ethyl. In other embodiments, R₁ is a group of the formula:

wherein:

-   -   R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),        substituted alkyl_((C≦6)), or a group of the formula:

-   -   wherein:        -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),            acyloxy_((C≦8)), or a substituted version of either of these            groups; and        -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted            version of either group;        -   q is 1, 2, or 3; and        -   r is 0, 1, 2, 3, or 4.

In some embodiments, R₇ is hydrogen. In other embodiments, R₇ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₇ may be alkyl_((C≦6)) suchas methyl or ethyl. In other embodiments, R₇ is

wherein:

-   -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),        acyloxy_((C≦8)), or a substituted version of either of these        groups; and    -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted        version of either group.

In some embodiments, R₉ is halo such as chloro or bromo. In otherembodiments, R₉ is hydroxy. In other embodiments, R₉ is alkoxy_((C≦8))or substituted alkoxy_((C≦8)). R₉ may be alkoxy_((C≦8)) such as methoxy.In some embodiments, R₉ is acyloxy_((C≦8)) or substitutedacyloxy_((C≦8)). R₉ may be acyloxy_((C≦8)) such as acetoxy orpivaloyloxy. In some embodiments, R₁₀ is alkyl_((C≦24)) or substitutedalkyl_((C≦24)) such as octyl, decyl, dodecyl, tetradecyl, hexadecyl, oroctadecyl. In other embodiments, R₁₀ is alkenyl_((C≦24)) or substitutedalkenyl_((C≦24)).

In some embodiments, R₈ is hydrogen. In other embodiments, R₈ isalkyl_((C≦6)) or substituted alkyl_((C≦6)). R₈ may be alkyl_((C≦6)) suchas methyl or ethyl. In other embodiments, R₈ is

wherein:

-   -   R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),        acyloxy_((C≦8)), or a substituted version of either of these        groups; and    -   R₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted        version of either group.

In some embodiments, R₉ is halo such as chloro or bromo. In otherembodiments, R₉ is hydroxy. In other embodiments, R₉ is alkoxy_((C≦8))or substituted alkoxy_((C≦8)). R₉ may be alkoxy_((C≦8)) such as methoxy.In some embodiments, R₉ is acyloxy_((C≦8)) or substitutedacyloxy_((C≦8)). R₉ may be acyloxy_((C≦8)) such as acetoxy orpivaloyloxy. In some embodiments, R₁₀ is alkyl_((C≦24)) or substitutedalkyl_((C≦24)) such as octyl, decyl, dodecyl, tetradecyl, hexadecyl, oroctadecyl. In other embodiments, R₁₀ is alkenyl_((C≦24)) or substitutedalkenyl_((C≦24)).

In some embodiments, q is 1 or 2. In one instance, q is 1. In anotheraspect, q is 2. In some embodiments, r is 1, 2, or 3. In one instance, ris 1. In another instance, r is 2. In another instance, r is 3. In someembodiments, the compounds are further defined as:

wherein:

-   -   R₁₁ is hydrogen, halo, hydroxy, or alkoxy_((C≦8)),        acyloxy_((C≦8)), or a substituted version of either of these        groups;        or a pharmaceutically acceptable salt thereof.

In still another aspect, the present disclosure provides compositionscomprising:

(A) a compound according to any one of claims 1-274; and

(B) a nucleic acid.

In some embodiments, the nucleic acid is a therapeutic nucleic acid. Insome embodiments, the nucleic acid is a short (small) interfering RNA(siRNA), a microRNA (miRNA), a messenger RNA (mRNA), a cluster regularlyinterspaced short palindromic repeats (CRISPR) RNA (crRNA), atrans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), atransfer RNA (tRNA), a plasmid DNA (pDNA), a double stranded DNA(dsDNA), a single stranded DNA (ssDNA), a single stranded RNA (ssRNA), adouble stranded RNA (dsRNA), a locked nucleic acid (LNA), a peptidenucleic acid (PNA), a miRNA mimic, or a anti-miRNA.

In some embodiments, the nucleic acid is a siRNA such as a siRNA usefulin the treatment of cancer. In other embodiments, the nucleic acid is atRNA such as a tRNA useful for correcting a nonsense mutation. In otherembodiments, the nucleic acid is an mRNA. In other embodiments, thenucleic acid is a sgRNA.

In some embodiments, the compositions further comprise a steroid orsteroid derivative. In some embodiments, the steroid or steroidderivative is a sterol such as cholesterol. In some embodiments, thecompositions further comprise a phospholipid. In some embodiments, thephospholipid is a phosphatidylcholine. In other embodiments, thephospholipid is distearoylphosphatidyl-choline. In some embodiments, thecompositions further comprise a PEG lipid. In some embodiments, the PEGlipid is a PEGylated diacylglycerol such as PEGylateddimyristoyl-sn-glycerol. In other embodiments, the PEG lipid is:

wherein:

-   -   n₁ is an integer from 1 to 250; and    -   n₂ and n₃ are each independently selected from 5, 6, 7, 8, 9,        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.

In some embodiments, n₁ is 5 to 100. In some embodiments, n₁ is 45. Insome embodiments, n₂ is 11, 12, 13, 14, 15, 16, or 17. In someembodiments, n₂ is 15. In some embodiments, n₃ is 11, 12, 13, 14, 15,16, or 17. In some embodiments, n₃ is 15.

In some embodiments, the compositions comprise a mole ratio of thecompound to the nucleic acid from about 5:1 to about 1000:1. In someembodiments, the mole ratio of the compound to the nucleic acid is fromabout 100:1 to about 1000:1. In some embodiments, the mole ratio isabout 166:1. In other embodiments, the mole ratio is from about 250:1 toabout 750:1 such as about 333:1 or about 666:1. In some embodiments, thecompositions comprise a ratio of the compound to the steroid or steroidderivative from about 1:1 to about 20:1 such as from about 1:1 to about6:1. In some embodiments, the ratio is from about 1.3:1. In someembodiments, the compositions comprise a ratio of the compound to thephospholipid is from about 1:1 to about 9:1 such as from about 2.5:1 toabout 7.5:1. In some embodiments, the ratio is about 5:1. In someembodiments, the compositions comprise a ratio of the compound to thePEG-lipid is from about 2.5:1 to about 100:1 such as from about 7.5:1 toabout 50:1. In some embodiments, the ratio is about 100:3. In someembodiments, the compositions comprise a ratio of the compound to thesteroid or steroid derivative to the phospholipid to the PEG lipid isfrom about 25:57:15:3 to about 75:19:5:1 such as about 50:38.5:10:1.5.

In some embodiments, the compositions further comprise apharmaceutically acceptable carrier. In some embodiments, thecompositions are formulated for administration: orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intramuscularly, intranasally, intraocularly,intrapericardially, intraperitoneally, intrapleurally,intraprostatically, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularly, intravitreally, liposomally, locally, mucosally,parenterally, rectally, subconjunctivally, subcutaneously, sublingually,topically, transbuccally, transdermally, vaginally, in crèmes, in lipidcompositions, via a catheter, via a lavage, via continuous infusion, viainfusion, via inhalation, via injection, via local delivery, or vialocalized perfusion. In some embodiments, the compositions areformulated for aerosol, intravenous, intraperitoneal, subcutaneous,topical, or oral administration. In other embodiments, the compositionsare formulated for injection such as for intraperitoneal injection orintravenous injection. In some embodiments, the compositions areformulated for inhalation.

In still yet another aspect, the present disclosure provides methods oftreating a disease or disorder in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of acompound or composition described herein.

In some embodiments, the disease or disorder is a genetic disease suchas a disease associated with a nonsense mutation. In some embodiments,the disease or disorder is cystic fibrosis, NGLY1 deficiency, Duchenemuscular dystrophy, thalassemia, Hurler syndrome, or Dravet syndrome.

In some embodiments, the disease or disorder is cystic fibrosis. In someembodiments, the methods further comprise a second therapeutic agent. Insome embodiments, the second therapeutic agent is another cysticfibrosis therapy. In some embodiments, the second therapeutic agent is atherapeutic agent useful for the management of cystic fibrosis. In someembodiments, the second therapeutic agent is an antibiotic, an agentuseful for maximizing organ function, or an agent useful for reducing oraltering the mucosal layer of the lungs. In some embodiments, the secondtherapeutic agent is an inhaled antibiotic, an oral antibiotic,ivacaftor, dornase alfa, hypertonic saline, denufosol, or acorticosteroid. In some embodiments, the methods further comprise asecond therapeutic modality. In some embodiments, the second therapeuticmodality is mechanical method of removing or reducing sputum.

In other embodiments, the disease or disorder is cancer. In someembodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia,melanoma, mesothelioma, multiple myeloma, or seminoma. In someembodiments, the cancer is of the bladder, blood, bone, brain, breast,central nervous system, cervix, colon, endometrium, esophagus, gallbladder, gastrointestinal tract, genitalia, genitourinary tract, head,kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa,ovary, pancreas, prostate, skin, spleen, small intestine, largeintestine, stomach, testicle, or thyroid. In some embodiments, thecancer is liver cancer, lung cancer, ovarian cancer, pancreatic cancer,breast cancer, leukemia cancer, or bone cancer. In some embodiments, thecancer is lung cancer or colorectal cancer. In some embodiments, thecancer has a nonsense mutation in a tumor suppressor gene such as amutation in the p53 gene. In some embodiments, the nonsense mutation isa mutation in the p53 gene in a lung cancer. In other embodiments, thenonsense mutation is in the APC gene such as a mutation in the APC genein a colorectal cancer. In other embodiments, the nonsense mutation isin the LKB1, ERCC3, WRN, BRCA2, IDH1, or ARID1A gene. In someembodiments, the cancer is a hepatitis B driven hepatocellularcarcinoma.

In some embodiments, the methods further comprise a second cancertherapy. In some embodiments, the second cancer therapy is a secondchemotherapeutic agent, an immunotherapy, a genetic therapy, or surgery.In some embodiments, the patient is a mammal such as a human. In someembodiments, the methods comprise administering the composition once. Inother embodiments, the methods comprise administering the compositiontwo or more times.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, amethod, composition, kit, or system that “comprises,” “has,” “contains,”or “includes” one or more recited steps or elements possesses thoserecited steps or elements, but is not limited to possessing only thosesteps or elements; it may possess (i.e., cover) elements or steps thatare not recited. Likewise, an element of a method, composition, kit, orsystem that “comprises,” “has,” “contains,” or “includes” one or morerecited features possesses those features, but is not limited topossessing only those features; it may possess features that are notrecited.

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this application, the term “average molecular weight” refersto the relationship between the number of moles of each polymer speciesand the molar mass of that species. In particular, each polymer moleculemay have different levels of polymerization and thus a different molarmass. The average molecular weight can be used to represent themolecular weight of a plurality of polymer molecules. Average molecularweight is typically synonymous with average molar mass. In particular,there are three major types of average molecular weight: number averagemolar mass, weight (mass) average molar mass, and Z-average molar mass.In the context of this application, unless otherwise specified, theaverage molecular weight represents either the number average molar massor weight average molar mass of the formula. In some embodiments, theaverage molecular weight is the number average molar mass. In someembodiments, the average molecular weight may be used to describe a PEGcomponent present in a lipid.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the description presentedherein.

FIG. 1 shows non-limiting examples of components of the cationicsulfonamide amino lipids of the present disclosure as a new class oflipids with properties enabling nucleic acid therapeutic delivery. Themodular design enabled systematic changes to the linker amine region(red), the headgroup amine (blue) and a functional sidearm (green) todetermine their relative contributions to biophysical properties. Stericinteractions around the quaternary amine, the number of lipid tails, andsidearm functionality were evaluated.

FIG. 2 shows an exemplary synthesis of CSALs based on the A1 linkeramine were performed from a common sulfobetaine zwitterionic precursor.

FIG. 3 shows CSAL nanoparticles are formed by the ethanol dilutionmethod, where combining a lipid mixture containing CSALs, cholesterol,DSPC, and PEG in ethanol at a mole ratio of 50:38.5:10:1.5 respectively,with a solution of siRNA in citrate phosphate buffer followed bydilution in PBS.

FIGS. 4A-4D show the biophysical characterization of A1-OAc CSAL, NPsshow structurally independent size ˜100 nm (FIG. 4A), siRNA bindingdecreases with increased headgroup linker length (FIG. 4B). Increasedcharge at a higher mole ratio indicates CSALs are present at thenanoparticle surface (FIG. 4C). Higher surface charge at pH 3 suggestchanges in protonation states of surface CSALs (FIG. 4D).

FIG. 5 shows siRNA delivery efficacy of A1-based CSALs was evaluated inHeLa luciferase reporter cells. NPs encapsulating siRNA against theluciferase reporter were dosed at 34 nM siRNA and incubated for 24 h.Relative luciferase activity (bars) and cell viability (dots) wereevaluated at different CSAL:siRNA molar ratio. C2Me headgroups andacetate sidearms at higher mole ratios showed greater efficacy.

FIG. 6 shows the design of new CSALs examined the effects of sterics onthe quaternary ammonium in the linker amine and in the head group amineThe C2Me head group shows the most activity. The four-tailed specieshighlights the importance tertiary amine content and hydrophobicity indelivery efficacy.

FIG. 7 shows uptake studies of CSAL-NPs in HeLa cells. Nanoparticleswere formulated with Cy5.5-labeled siRNA (red) at 333:1 CSAL:siRNA moleratio and incubated at 17.1 nM siRNA for 24 h. Cells were counterstainedwith DAPI (blue) overlayed with siRNA signal.

FIG. 8 shows the biodistribution of CSAL NPs in vivo. Cy5.5-labeledsiRNA was encapsulated in CSAL NPs and injected systemically (1 mg/kgsiRNA dose). A1OAcC2Me and A3OAcC2Me localize to lung after IVadministration, A3OAcC2Me localizes to liver after IP administration.The effect of total lipid:siRNA weight ratio on A3OAcC2Mebiodistribution was examined. All weight ratios resulted in lungaccumulation at 2 h, while significant clearance to kidney was observedafter 24 h at lower weight ratio suggesting better stability at higherweight ratio.

FIG. 9 shows non-limiting examples of some of the modular componentsfrom which the zwitterionic amino lipids may be prepared. The greenmolecules represent the electrophilic amine with the appropriatecationic amine group, red represents the core polyamine, and blue is thehydrophobic tails.

FIG. 10 shows the ¹H NMR spectra of the zwitterionic electrophilecomponent and an exemplary synthesis of that component.

FIG. 11 shows the ¹H NMR spectra of the zwitterionic electrophilecomponent coupled to the poly amine and an exemplary synthesis of thisjoint component.

FIG. 12 shows the variety of zwitterionic components with differentpolyamines

FIG. 13 shows the reaction of the components described in FIG. 12 with avariety of different hydrophobic components.

FIG. 14 shows the resultant zwitterionic amino lipids (ZALs) from thereaction shown in FIG. 11.

FIGS. 15A-15C show the HPLC traces of ZA3-Ep10 (FIG. 15A), 12SBAmO10(FIG. 15B), and ZA1-Am10 (FIG. 15C).

FIG. 16 shows the formation of nanoparticles comprising siRNA and ZALsin the presence of one or more helper lipids such as cholesterol.

FIG. 17 shows that ZALs in the presence of cholesterol are able toencapsulate and bind siRNA.

FIG. 18 shows the luciferase activity as a function of in vitro siRNAdelivery in HeLa cells. Darker colors represent lower activity ofluciferase and higher siRNA delivery.

FIG. 19 shows the activity as a percentage of untreated cells (bars) andcell viability (dots) with the different lipid types noted by the colorof the bars.

FIGS. 20A & 20B show the activity as a percentage of untreated cells(bar) and cell viability (dots) with different length and core amine forepoxide based lipids (FIG. 20A) and acrylate based lipids (FIG. 20B).

FIG. 21 shows that ZAL nanoparticles containing tRNA are internalizedinto Calu6 cells. These particles have been shown to taken up bymultiple different cell lines.

FIG. 22 shows restoration of p53 synthesis after delivery of the tRNAusing different delivery composition.

FIG. 23 shows distribution of a cationic sulfonamide and a zwitterionicamino lipid to different organs in vivo.

FIG. 24 shows delivery of tRNA into primary HBE cells using azwitterionic amino lipid.

FIG. 25 shows the effects of combining multiple different nucleic acidmolecules in a single nanoparticle. Co-delivery of Cas9 mRNA and sgRNAagainst luciferase to HeLa-Luc cells was tested with an RNA dose of 100ng mRNA per well and 50 ng sgRNA per well in 200 μL DMEM 5% FBS. “Sameparticle” samples are nanoparticles packaged with both mRNA and sgRNA inthe same nanoparticle formulation by co-diluting the mRNA and sgRNAtogether in acidic buffer prior to the addition of the lipids duringformulaiton. “Diff particle” samples are are the mRNA-LNP and sgRNA-LNPprepared as separate nanoparticles, but added at the same time to thesame wells. “Staggered” samples are mRNA-ZAL nanoparticles areformulated and added 16 h prior to sgRNA-ZAL nanoparticles. As anegative cotrol “sgLuc only 43 h,” nanoparticles with sgRNA againstluciferase were added in the absence of Cas9 mRNA. Samples were read out43 h after the initial transfection of mRNA alone or mRNA+sgRNA.

FIG. 26 shows co-delivery of Cas9 mRNA and single-guide RNA by ZA3-Ep10nanoparticles against luciferase to A549-luc cells.

FIG. 27 shows co-delivery of Cas9 mRNA and single-guide RNA by ZA3-Ep10nanoparticles against luciferase to HeLa-luc cells.

FIG. 28 shows a dose response distribution for ZAL compositions atparticular weigh ratio of sgRNA. These dose response distribution wascarried out with ZA3-Ep10 NPs in HeLa-Luc-Cas9 cells. The compositioncomprised 50:38.5:2 ZAL:Cholesterol:PEG-lipid, at 20:1, 10:1, 5:1ZAL:sgRNA weight ratio in nanoparticle formulations. The composition wasincubated for 48 hours. 50 ng siLuc was used as the siRNA positivecontrol.

FIG. 29 shows ex vivo imaging of BALB-c-Nu mice which have been injectedeither intravenously or intraperitoneally with 1 mg/kg Luc mRNA. Themice were imaged 24 hours post injection. These images show the organlocalization of nanoparticles.

FIG. 30 shows the dose dependent activity of luciferase siRNA whendelivered using CSALs at two different ratios of nucleic acid tonanoparticle ratios and two different CSALs.

FIG. 31 shows the localization of the CSALs in A549-luc cells with 34 nMsiRNA. The image is taken after 24 hours of incubation. The scale bar atthe bottom right corner of the images is 40 μm.

FIG. 32 shows the localization of A3OAcC2Me nanoparticles (50 CSAL:38.5Cholesterol: 10 DSPC: 1.5 PEG mol ratios in lipid mix, 30:1 totallipid:siRNA weight ratio) in A549-luc xenografts in Balb-c nude mice.The nanoparticles were injected intratumoral injection with 1 mg/kgsiRNA using bioluminescence imaging after 24 h using IVIS with IPluciferin injection. Ex vivo analysis (48 h) included sacrificing theanimals and tissue collected and frozen on dry ice. Then, the tissue washomogenized by a tissue homogenizer followed by tip sonication in 1×lysis buffer (Promega) and supplemented with protease inhibitor(Pierce). The samples were normalized by total mass tumor tissue(N=4+/−S.E.M.).

FIG. 33 shows the binding and particle size from the compositions usedin tRNA delivery in Calu6 cells.

FIG. 34 shows a gel showing that both ZAL and CSAL nanoparticles enablethe delivery of suppressor tRNA which result in the restoration of p53expression.

FIG. 35 shows the uptake of a variety of different ZALs withfluorescently labeled tRNA nanoparticle formations in Calu6 cells.

FIG. 36 shows the ¹H NMR spectrum of 6 different ZALs with amino ZAs.

FIG. 37 shows the characterization of the purified ZA3-Ep10 ZALincluding ELDS, mass spectroscopy, and ¹³C NMR.

FIG. 38 shows the characterization of CSAL A3OAcC2Me via massspectroscopy and ¹³C NMR.

FIG. 39 shows alternative synthesis methods for preparation of the ZALs.

FIG. 40 shows alternative synthesis methods for the preparation of theCSALs and ZALs.

FIGS. 41A-41D show ¹H NMR spectra of CSALs: A1OAcC2Me (FIG. 41A),A1OAcC3Me (FIG. 41B), A1OAcC4Me (FIG. 41C), and ZA (FIG. 41D).

FIGS. 42A-42F show ¹¹H NMR spectra of ZALs: ZA (FIG. 42A), ZA3-Ep10(FIG. 42B), A1-OH (FIG. 42C), A3-OH (FIG. 42D), A3-OAc (FIG. 42E), andA1-OPiv (FIG. 42F).

FIGS. 43A-43C show Cas9 expression was validated in HeLa-Luc-Cas9 cellsby western blot. (FIG. 43A) Blotting with tx-FLAG antibody in the poolof cells after Blasticidin S selection. (FIG. 43B) Luciferase expressionof single cell clones as evaluated by the One-Glo assay (5,000 cells, 48h growth). (FIG. 43C) Cas9 expression of single cell clone 2 ofHeLa-Luc-Cas9 blotted with α-Cas9.

FIG. 44 shows the evaluation of panel of single guide RNAs againstluciferase using commercial reagent (LF3000) transfection of plasmid DNAencoding sgRNA and Cas9 protein reveals sgLuc5 as the most potent sgRNAsequence for silencing luciferase in unsorted. HeLa-Linc cells. Valuesare normalized to non-targeting sgRNA control and plotted asmean+/−standard deviation (N=4).

FIG. 45 shows lead ZALs identified from the snRNA screen were evaluatedfor sgRNA delivery to HeLa-Luc-Cas9 cells. ZNPs were formulated at50:38.5:1 (ZAL:cholesterol:PEG-lipid molar ratios) n the lipid mix and20:1 ZAL:sgRNA weight ratio. sgRNA was administered at both 14.7 nM and7.4 nM for 48 h. ZA3-Ep10 emerged as the most highly potent (>95%luciferase silencing). Viability (dots) and relative luciferase activity(bars) were determined relative to untreated cells (N=4+/−standarddeviation).

FIG. 46 shows magnification of the early time points of the kineticcurve of luciferase silencing comparing sgRNA versus siRNA by ZA3-Ep10ZNPs shows that siRNA silencing is much faster than sgRNA editing.

FIG. 47 shows the relative viability of ZNP edited HeLa-Luc-Cas9 cells(sgLuc) versus unedited cells (sgCtrl) shows similar growth rates by theCell-Titer Glo assay when normalized to untreated cells (N=5+/−S.E.M.)

FIG. 48 shows the optimization of ZA3-Ep10 ZNPs for sgRNA delivery wasexplored by tuning the PEG content of the formulation (2%, 1%, and 0.5%)and the ZAL:sgRNA weight ratio (20:1, 10:1, 7.5:1 5:1). All formulationswere potent for sgLuc delivery at 7.4 nM, 48 h incubation, while 7.5:1weight ratio and 0.5% PEG showed the best luciferase editing.

FIG. 49 shows the optimization of the ZA3-Ep10 ZNPs for mRNA deliverywas performed in IGROV1 cells. The weight ratio of the ZAL:mRNA was setat 20:1, 10:1, 7.5:1 and 5:1. The lipid mix was prepared with a relativemolar ratio of 50:38.5:n, ZAL:cholesterol:PEG-lipid, where n=5, 2, 1 or0.5. Cells were treated in 96-well plates with 100 ng mRNA and incubatedfor the indicted time (18 h light gray, 26 h gray, 45 h dark gray) priorto evaluation of cell viability (dots) and luciferase expression (bars)using the One-Glo+Tox assay. Cell viability was determined compared tountreated cells and luminescence was normalized to viability todetermine relative luminescence. Values are plotted as a mean+/−standarddeviation, N=4.

FIG. 50 shows the effect of PEG lipid composition of ZA3-Ep10 Luc mRNANPs formulated for in vivo assays. The ZAL:cholesterol ratio was fixedat 50:38.5 molar ratio while PEG-lipid was included at the indicatedpercentage. As expected increased PEG leads to smaller particle size,but poorer expression of mRNA.

FIG. 51 shows that comparing the RNA encapsulation, nanoparticle size,and delivery efficacy of ZA3-Ep10 and a cationic structural analogue(A3-Ep14, also referred to as C14-110 in the literature (Love et al.,2010)), which is known to deliver small RNA. The ZNP or LNP formulationwas fixed at 7.5:1 weight ratio ZAL or Cationic analogue to RNA. Thelipid mixture for the NPs was 50:38.5:0.5 ZAL or cationic analogue:cholesterol: PEG-lipid, while for the A3-Ep14 NPs the zwitterionicphospholipid was titrated from 0 to 50% in the lipid mix. Thenanoparticles were formulated by manual mixing using the in vitroformulation protocol. RNA binding was determined by the Ribogreen assay(N=3+/−standard deviation), while nanoparticle size was determined bydynamic light scattering (N=3+/−standard deviation). Luciferasesilencing or editing of siLuc and sgLuc NPs was assayed in HeLa-Luc-Cas9cells (7.35 nM sgRNA, 17.9 nM siRNA), while luciferase expression by LucmRNA NPs was evaluated in IGROV1 cells (0.77 nM mRNA). Cells were assaysafter 40 h incubation time by the One-Glo+Tox assay and plotted withviability (dots) and luciferase expression (bars) as mean+/−standarddeviation (N=4).

FIG. 52 shows bioluminescence imaging shows that in vivo expression ofluciferase after Luc-mRNA administration by i.v. injection correlateswith in vitro activity. Mice were injected with 1 mg/kg Luc mRNA andimaged 24 h after treatment. An untreated mouse was used as a negativecontrol. The top right panel shows the ex vivo expression of the animalshown in FIG. 65E.

FIGS. 53A & 53B show quantitation of the ex vivo images by ROI analysis.(FIG. 53A) Quantitation of the athymic nude mice images shown in FIG. 52(top) and (FIG. 53B) quantitation images of the images in FIG. 51(bottom, NSG) and FIG. 65F (C57BL/6). A minimum of 5 ROIs per organ wasmeasured and plotted as mean+/−S.E.M.

FIG. 54 shows co-delivery of Cas9 mRNA and sgLuc leads to editing instaged delivery at 2 μg per well Cas9 mRNA and 1 μg sgLuc in a 6-wellplate in both A549-Luc and HeLa-Luc. Meanwhile, unguided Cas9,Cas9-sgCtrl, or sgLuc alone do not show edited bands. The expectedgenomic DNA amplicon was 510 bp while the expected cut bands indicatingediting are 233 bp and 277 bp (arrows).

FIG. 55 shows control ZNPs (Cas9+sgCtrl, unguided Cas9, sgLuc only andsgCtrl only) did not show editing of luciferase target in A549-Luccells. Staged co-delivery shows editing with sgLuc under similarconditons with 2:1 Cas9 mRNA:sgLuc wr.

FIG. 56 shows the encapsulation of Cas9 mRNA and sgRNA in co-deliveryZNPs. ZAL: total RNA was fixed at 7.5:1, with a lipid mixture of50:38.5:0.5 ZA3-Ep10: cholesterol: PEG-lipid. Data are plotted asmean+/−standard deviation (N=4).

FIG. 57 shows particle properties of in vivo administered ZNPsencapsulating Cas9 mRNA and sgRNA. For size and zeta potentialmeasurements, N=5 for RNA encapsulation N=4. Data are plotted asmean+/−standard deviation.

FIG. 58 shows the Cre recombinase AAV positive control demonstratesexpression of tdTomato in liver ex vivo at the whole organ level and incells from tissue sections.

FIG. 59 shows the delivery of ZA3-Ep10 ZNPs encapsulating Cas9 mRNA andsgCtrl does not show any tdTomato positive cells in sectioned tissueslides.

FIG. 60 shows the measurement of animal body weight after systemicadministration of ZA3-Ep10 ZNPs encapsulating Cas9 mRNA and sgRNA at 5mg/kg total RNA dose.

FIG. 61 shows the quantification of tdTomato positive hepatocytes inanimals treated with ZNPs as determined by flow cytometry of isolatedprimary hepatocytes. The left panel shows representative plots ofsamples from an untreated LSL-tdTO mouse and a ZNP-Cas9 mRNA-sgLoxPtreated mouse. Mouse 1 and mouse 2 were treated at 2 mg/kg total RNA 2times on consecutive days, while mouse 3 received a single dose at 5mg/kg total RNA and all animals were harvested ˜1 week after ZNPadministration. Each sample was run four times and values are plotted asmean+/−standard deviation.

FIG. 62 shows that a ZNP treated tdTomato mouse shows significantfluorescent signal in the liver and kidneys 2 months after editing byZA3-Ep10 ZNPs encapsulating Cas9 mRNA and sgLoxP (5 mg/kg).

FIGS. 63A-63D shows that ZNPs enable permanent CRISPR/Cas-mediated DNAediting. (FIG. 63A) Sequence specific silencing of luciferase by siRNA(9 nM) and editing by sgRNA (7 nM) in HeLa-Luc-Cas9 cells. N=4±stdev,****p<0.0001 (FIG. 63B) Kinetically, silencing with siRNA is transientwhile sgRNA delivery results in permanent loss of luciferase signalafter 2 days. (FIG. 63C) Sequence specific editing of luciferase wasconfirmed by the Surveyor assay. (FIG. 63D) The chemical structure ofZA3-Ep10.

FIG. 64 shows ZALs were designed to increase molecular interactions withlonger RNAs by combining the chemical and structural roles ofzwitterionic lipids and cationic lipids into a single lipid compound.High efficiency reactions provided access to a library of unique chargeunbalanced lipids.

FIGS. 65A-65F show ZNPs enable delivery of long RNAs both in vitro andin vivo. (FIG. 65A) ZA3-Ep10 ZNPs (ZAL:cholesterol:PEG-lipid=100:77:1(mol); ZAL:RNA=7.5:1 (wt)) are uniform for both sgRNA and mRNA. (FIG.65B) ZA3-Ep10 sgRNA ZNPs show dose-responsive Luc editing inHeLa-Luc-Cas9 cells. ZA3-Ep10 ZNPs can also deliver (FIG. 65C) mCherrymRNA (18 h) and (FIG. 65D) luciferase mRNA (24 h) to IGROV1 cells. (FIG.65E) In vivo luciferase expression was achieved by systemic i.v.administration of ZA3-Ep10 Luc mRNA ZNPs (24 h). Bioluminescence imagingboth in vivo (FIG. 65E, athymic nude mice, 1 mg/kg) and ex vivo (FIG.65F, C57BL/6 mice, 4 mg/kg) revealed expression of luciferase in liver,lung and spleen tissue.

FIGS. 66A-66D show ZNPs enable co-delivery of Cas9 mRNA and sgRNA forCRISPR/Cas editing. (FIG. 66A) The kinetics of mRNA and proteinexpression after ZNP delivery of Cas9 mRNA (0.48 ng/mL mRNA) to A549-Luccells. Cas9 mRNA levels (A light gray curve) and protein expression (Ablack curve, FIG. 66B) were measured over time. (FIG. 66C) ZNPs enabledose responsive expression of Cas9, detectable as low as 0.05 μg/mLdelivered mRNA. (FIG. 66D) Surveyor confirmed editing of the luciferasetarget at mRNA:sgRNA ratios of 3:1 or higher (wt). Co-delivery of Cas9mRNA and sgCtrl showed no editing (FIG. 55).

FIGS. 67A-647C show ZNPs enabled non-viral CRISPR/Cas editing in vivo.(FIG. 67A) Schematic representation shows that co-delivery of Cas9 mRNAand sgLoxP deletes the stop cassette and activates downstream tdTomatoprotein. (FIG. 67B) After administration of ZNPs encapsulating Cas9mRNA:sgRNA (4:1, wt) at 5 mg/kg total RNA, tdTomato fluorescence wasdetected in the liver and kidney upon whole organ ex vivo imaging. (FIG.67C) Confocal fluorescence microscopy of tissue sections showed tdTomatopositive cells in liver, lung, and kidneys. Scale bars=50 μm).

FIG. 68 shows the ¹H NMR of ZA3-Ep10-OAc (top) relative to ZA3-Ep10(bottom). The spectrum at the top shows the presence of the methyl groupon the acetyl moiety (circle) at about 2 ppm.

FIG. 69 show the ¹H NMR spectra for the glycidic ester of olelyl.

FIGS. 70A-70O show the mass spectra of modified ZAL compounds: ZA3-GE8(FIG. 70A), ZA3-GE12 (FIG. 70B), ZA3-Ac-oleyl (FIG. 70C), ZA3-Ep10-OAc(FIG. 70D), ZA3-Ep10-OPiv (FIG. 70E), ZA3-Ep10-alkyl (FIG. 70F),ZA3-Ep10-Octanoate (FIG. 70G), 4A2SBAm (FIG. 70H), 4A4SBAm (FIG. 70I),SBAm-C3Me (FIG. 70J), SBAm-C2Et (FIG. 70K), SBAm-C3Et (FIG. 70L),CBAm-C2Me (FIG. 70M), C4SBAm-C2Me (FIG. 70N), and iPCAm-C2Me (FIG. 70O).

FIGS. 71A-E show the ¹H NMR spectra of the modified ZAL compounds:ZA3-Ac-oleyl (FIG. 71A), GE12 (FIG. 71B), ZA3-GE12 (FIG. 71C), ZA3-Ep10-OAc (FIG. 71D), and ZA3-Ep10-OPiv (FIG. 71E).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure provides amino lipid composition containing oneor more sulfonic acid or a sulfonic acid derivative such as asulfonamide. These compounds may be combined with one or more helperlipids to form nanoparticles in aqueous solution which may be used totransport nucleic acid based therapeutic agents. In some embodiments,the present compositions may be used to transport siRNA, sgRNA, mRNA, ortRNA therapeutics to treating a disease or disorder such as cancer,cystic fibrosis, or other genetic disorders.

A. DEFINTIONS

The compounds (also described as an amino lipid, a compound, or acompound of the present disclosure herein) provided by the presentdisclosure are shown, for example, above in the summary section and inthe claims below. They may be made using the methods outlined in theExamples section. These methods can be further modified and optimizedusing the principles and techniques of organic chemistry as applied by aperson skilled in the art. Such principles and techniques are taught,for example, in March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure (2007), which is incorporated by referenceherein.

Compounds of the present disclosure may contain one or moreasymmetrically-substituted carbon or nitrogen atoms, and may be isolatedin optically active or racemic form. Thus, all chiral, diastereomeric,racemic form, epimeric form, and all geometric isomeric forms of achemical formula are intended, unless the specific stereochemistry orisomeric form is specifically indicated. Compounds may occur asracemates and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. In some embodiments, a singlediastereomer is obtained. The chiral centers of the compounds of thepresent disclosure can have the S or the R configuration.

Chemical formulas used to represent compounds of the disclosure willtypically only show one of possibly several different tautomers. Forexample, many types of ketone groups are known to exist in equilibriumwith corresponding enol groups. Similarly, many types of imine groupsexist in equilibrium with enamine groups. Regardless of which tautomeris depicted for a given compound, and regardless of which one is mostprevalent, all tautomers of a given chemical formula are intended.

Compounds of the present disclosure may also have the advantage thatthey may be more efficacious than, be less toxic than, be longer actingthan, be more potent than, produce fewer side effects than, be moreeasily absorbed than, and/or have a better pharmacokinetic profile(e.g., higher oral bioavailability and/or lower clearance) than, and/orhave other useful pharmacological, physical, or chemical propertiesover, compounds known in the prior art, whether for use in theindications stated herein or otherwise.

In addition, atoms making up the compounds of the present disclosure areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C.

It should be recognized that the particular anion or cation forming apart of any salt form of a compound provided herein is not critical, solong as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and theirmethods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (2002), which is incorporatedherein by reference.

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means ═NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof; “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “ - - - ”represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, theformula

includes

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“—”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it covers all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularly across a bond (e.g.

or methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the chemical groups and compound classes, the number of carbon atomsin the group or class is as indicated as follows: “Cn” defines the exactnumber (n) of carbon atoms in the group/class. “C≦n” defines the maximumnumber (n) of carbon atoms that can be in the group/class, with theminimum number as small as possible for the group/class in question,e.g., it is understood that the minimum number of carbon atoms in thegroup “alkenyl_((C≦8))” or the class “alkene_((C≦8))” is two. Comparewith “alkoxy_((C≦10))”, which designates alkoxy groups having from 1 to10 carbon atoms. “Cn-n′” defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Thus, “alkyl_((C2-10))” designatesthose alkyl groups having from 2 to 10 carbon atoms. These carbon numberindicators may precede or follow the chemical groups or class itmodifies and it may or may not be enclosed in parenthesis, withoutsignifying any change in meaning. Thus, the terms “C5 olefin”,“C5-olefin”, “olefin_((C5))”, and “olefin_(C5)” are all synonymous. Whenany of the chemical groups or compound classes defined herein ismodified by the term “substituted”, any carbon atom(s) in a moietyreplacing a hydrogen atom is not counted. Thus methoxyhexyl is anexample of a substituted alkyl_((C1-6)).

The term “saturated” when used to modify a compound or chemical groupmeans the compound or chemical group has no carbon-carbon double and nocarbon-carbon triple bonds, except as noted below. When the term is usedto modify an atom, it means that the atom is not part of any double ortriple bond. In the case of substituted versions of saturated groups,one or more carbon oxygen double bond or a carbon nitrogen double bondmay be present. And when such a bond is present, then carbon-carbondouble bonds that may occur as part of keto-enol tautomerism orimine/enamine tautomerism are not precluded. When the term “saturated”is used to modify a solution of a substance, it means that no more ofthat substance can dissolve in that solution.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound or chemical group so modified is an acyclicor cyclic, but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by singlecarbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or morecarbon-carbon double bonds (alkenes/alkenyl) or with one or morecarbon-carbon triple bonds (alkynes/alkynyl).

The term “aromatic” when used to modify a compound or a chemical grouprefers to a planar unsaturated ring of atoms with 4n+2 electrons in afully conjugated cyclic π system.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂ (i-Pr, ^(i)Pr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃ (tent-butyl, t-butyl, t-Bu or ^(t)Bu), and—CH₂C(CH₃)₃ (neo-pentyl) are non-limiting examples of alkyl groups. Theterm “alkanediyl” when used without the “substituted” modifier refers toa divalent saturated aliphatic group, with one or two saturated carbonatom(s) as the point(s) of attachment, a linear or branched acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, and —CH₂CH₂CH₂— are non-limiting examples of alkanediylgroups. The term “alkylidene” when used without the “substituted”modifier refers to the divalent group ═CRR′ in which R and R′ areindependently hydrogen or alkyl. Non-limiting examples of alkylidenegroups include: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. An “alkane” refers tothe class of compounds having the formula H—R, wherein R is alkyl asthis term is defined above. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂,—C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.The following groups are non-limiting examples of substituted alkylgroups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH, —CH₂C(O)OCH₃,—CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂N(CH₃)₂,and —CH₂CH₂Cl. The term “haloalkyl” is a subset of substituted alkyl, inwhich the hydrogen atom replacement is limited to halo (i.e. —F, —Cl,—Br, or —I) such that no other atoms aside from carbon, hydrogen andhalogen are present. The group, —CH₂Cl is a non-limiting example of ahaloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, inwhich the hydrogen atom replacement is limited to fluoro such that noother atoms aside from carbon, hydrogen and fluorine are present. Thegroups —CH₂F, —CF₃, and —CH₂CF₃ are non-limiting examples of fluoroalkylgroups.

The term “cycloalkyl” when used without the “substituted” modifierrefers to a monovalent saturated aliphatic group with a carbon atom asthe point of attachment, said carbon atom forming part of one or morenon-aromatic ring structures, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. Non-limiting examplesinclude: —CH(CH₂)₂ (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl(Cy). The term “cycloalkanediyl” when used without the “substituted”modifier refers to a divalent saturated aliphatic group with two carbonatoms as points of attachment, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. The group

is a non-limiting example of cycloalkanediyl group. A “cycloalkane”refers to the class of compounds having the formula H—R, wherein R iscycloalkyl as this term is defined above. When any of these terms isused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, acyclic structure, at leastone nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples include: —CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂(allyl), —CH₂CH═CHCH₃, and —CH═CHCH═CH₂. The term “alkenediyl” when usedwithout the “substituted” modifier refers to a divalent unsaturatedaliphatic group, with two carbon atoms as points of attachment, a linearor branched, a linear or branched acyclic structure, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds,and no atoms other than carbon and hydrogen. The groups —CH═CH—,—CH═C(CH₃)CH₂, —CH═CHCH₂, and —CH₂CH═CHCH₂are non-limiting examples ofalkenediyl groups. It is noted that while the alkenediyl group isaliphatic, once connected at both ends, this group is not precluded fromforming part of an aromatic structure. The terms “alkene” and “olefin”are synonymous and refer to the class of compounds having the formulaH—R, wherein R is alkenyl as this term is defined above. Similarly theterms “terminal alkene” and “α-olefin” are synonymous and refer to analkene having just one carbon-carbon double bond, wherein that bond ispart of a vinyl group at an end of the molecule. When any of these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —CH═CHF, —CH═CHCl and —CH═CHBr arenon-limiting examples of substituted alkenyl groups.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched acyclic structure, at leastone carbon-carbon triple bond, and no atoms other than carbon andhydrogen. As used herein, the term alkynyl does not preclude thepresence of one or more non-aromatic carbon-carbon double bonds. Thegroups —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃ are non-limiting examples ofalkynyl groups. An “alkyne” refers to the class of compounds having theformula H—R, wherein R is alkynyl. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, anda monovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl, aryl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. If more than one ring is present, the rings may be fused orunfused. Unfused rings may be connected via one or more of thefollowing: a covalent bond, alkanediyl, or alkenediyl groups (carbonnumber limitation permitting). Non-limiting examples of arenediyl groupsinclude:

An “arene” refers to the class of compounds having the formula H—R,wherein R is aryl as that term is defined above. Benzene and toluene arenon-limiting examples of arenes. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group alkanediylaryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and2-phenyl-ethyl. When the term aralkyl is used with the “substituted”modifier one or more hydrogen atom from the alkanediyl and/or the arylgroup has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than onering is present, the rings may be fused or unfused. As used herein, theterm does not preclude the presence of one or more alkyl, aryl, and/oraralkyl groups (carbon number limitation permitting) attached to thearomatic ring or aromatic ring system. Non-limiting examples ofheteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im),isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl(pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl,quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.The term “heteroarenediyl” when used without the “substituted” modifierrefers to an divalent aromatic group, with two aromatic carbon atoms,two aromatic nitrogen atoms, or one aromatic carbon atom and onearomatic nitrogen atom as the two points of attachment, said atomsforming part of one or more aromatic ring structure(s) wherein at leastone of the ring atoms is nitrogen, oxygen or sulfur, and wherein thedivalent group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than onering is present, the rings may be fused or unfused. Unfused rings may beconnected via one or more of the following: a covalent bond, alkanediyl,or alkenediyl groups (carbon number limitation permitting). As usedherein, the term does not preclude the presence of one or more alkyl,aryl, and/or aralkyl groups (carbon number limitation permitting)attached to the aromatic ring or aromatic ring system. Non-limitingexamples of heteroarenediyl groups include:

The term “N-heteroaryl” refers to a heteroaryl group with a nitrogenatom as the point of attachment. A “heteroarene” refers to the class ofcompounds having the formula H—R, wherein R is heteroaryl. Pyridine andquinoline are non-limiting examples of heteroarenes. When these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present,the rings may be fused or unfused. As used herein, the term does notpreclude the presence of one or more alkyl groups (carbon numberlimitation permitting) attached to the ring or ring system. Also, theterm does not preclude the presence of one or more double bonds in thering or ring system, provided that the resulting group remainsnon-aromatic. Non-limiting examples of heterocycloalkyl groups includeaziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl,morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl,tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term“heterocycloalkanediyl” when used without the “substituted” modifierrefers to an divalent cyclic group, with two carbon atoms, two nitrogenatoms, or one carbon atom and one nitrogen atom as the two points ofattachment, said atoms forming part of one or more ring structure(s)wherein at least one of the ring atoms is nitrogen, oxygen or sulfur,and wherein the divalent group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present,the rings may be fused or unfused. Unfused rings may be connected viaone or more of the following: a covalent bond, alkanediyl, or alkenediylgroups (carbon number limitation permitting). As used herein, the termdoes not preclude the presence of one or more alkyl groups (carbonnumber limitation permitting) attached to the ring or ring system. Also,the term does not preclude the presence of one or more double bonds inthe ring or ring system, provided that the resulting group remainsnon-aromatic. Non-limiting examples of heterocycloalkanediyl groupsinclude:

The term “N-heterocycloalkyl” refers to a heterocycloalkyl group with anitrogen atom as the point of attachment. N-pyrrolidinyl is an exampleof such a group. When these terms are used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl,aryl, aralkyl or heteroaryl, as those terms are defined above. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅,—C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl”is defined in an analogous manner, except that the oxygen atom of thegroup —C(O)R has been replaced with a sulfur atom, —C(S)R. The term“aldehyde” corresponds to an alkane, as defined above, wherein at leastone of the hydrogen atoms has been replaced with a —CHO group. When anyof these terms are used with the “substituted” modifier one or morehydrogen atom (including a hydrogen atom directly attached to the carbonatom of the carbonyl or thiocarbonyl group, if any) has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl),—CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and—CON(CH₃)₂, are non-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples include: —OCH₃ (methoxy), —OCH₂CH₃ (ethoxy),—OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), —OC(CH₃)₃ (tert-butoxy),—OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl. The terms “cycloalkoxy”,“alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is cycloalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl,respectively. The term “alkylthio” and “acylthio” when used without the“substituted” modifier refers to the group —SR, in which R is an alkyland acyl, respectively. The term “alcohol” corresponds to an alkane, asdefined above, wherein at least one of the hydrogen atoms has beenreplaced with a hydroxy group. The term “ether” corresponds to analkane, as defined above, wherein at least one of the hydrogen atoms hasbeen replaced with an alkoxy group. When any of these terms is used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples include: —NHCH₃ and —NHCH₂CH₃. Theterm “dialkylamino” when used without the “substituted” modifier refersto the group —NRR′, in which R and R′ can be the same or different alkylgroups, or R and R′ can be taken together to represent an alkanediyl.Non-limiting examples of dialkylamino groups include: —N(CH₃)₂ and—N(CH₃)(CH₂CH₃). The terms “cycloalkylamino”, “alkenylamino”,“alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”,“heterocycloalkylamino”, “alkoxyamino”, and “alkylsulfonylamino” whenused without the “substituted” modifier, refers to groups, defined as—NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl,heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. Anon-limiting example of an arylamino group is —NHC₆H₅. The term “amido”(acylamino), when used without the “substituted” modifier, refers to thegroup —NHR, in which R is acyl, as that term is defined above. Anon-limiting example of an amido group is —NHC(O)CH₃. The term“alkylimino” when used without the “substituted” modifier refers to thedivalent group ═NR, in which R is an alkyl, as that term is definedabove. When any of these terms is used with the “substituted” modifierone or more hydrogen atom attached to a carbon atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent disclosure which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this disclosure is not critical, so longas the salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2^(n), where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≦15%, morepreferably ≦10%, even more preferably ≦5%, or most preferably ≦1% ofanother stereoisomer(s).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in anyreference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the disclosure in terms such thatone of ordinary skill can appreciate the scope and practice the presentdisclosure.

B. AMINO LIPIDS

In some aspects, the present disclosure provides one or more amino lipidcompounds containing two or more nitrogen atoms and a sulfonic acid orsulfonic acid derivative such as a sulfonamide In some embodiments, oneclass of amino lipids is a cationic sulfonamide amino lipid whichcontains two or more nitrogen atoms wherein at least one of the nitrogenatoms is an amine which is protonated at physiological pH, two or morelipid groups, and a sulfonamide group. This class of amino lipidscontains two or more lipid groups wherein the lipid group is a C6-C24aliphatic group including alkyl, alkenyl, alkynyl groups or asubstituted version of these groups. These lipid groups are connected tothe rest of the amino lipid groups through an ester, an amide, or anepoxide. In some embodiments, the lipid group is a C6-C24 alkyl orsubstituted alkyl group.

In other embodiments, another class of amino lipids described herein isa zwitterionic amino lipid which contains two or more nitrogen atomswherein at least one of the nitrogen atoms is a quaternary ammoniumatom, a negatively charged group, and two or more lipid groups. Thenegatively charged group may be a phosphonic acid group or a sulfonicgroup. In some embodiments, the negatively charged group is a sulfonicgroup. As described above, the lipid groups are a C6-C24 aliphatic groupincluding alkyl, alkenyl, alkynyl groups or a substituted version ofthese groups. These lipid groups are connected to the rest of the aminolipid groups through an ester, an amide, or an epoxide. In someembodiments, the lipid group is a C6-C24 alkyl or substituted alkylgroup.

In some embodiments, the present composition comprises a ratio of thecompound or amino lipids to the nucleic acid from about 1:1 to about1500:1 or from about 5:1 to about 1000:1. The ratio may be from about100:1-1000:1 or from about 250:1 to about 750:1 such as a ratio of about166:1, 333:1, or 666:1. In some embodiments, the ratio is from about1:1, 5:1, 25:1, 50:1, 75:1, 100:1, 200:1, 300:1, 350:1, 400:1, 500:1,600:1, 650:1, 700:1, 750:1, 800:1, 900:1, to about 1000:1, or any rangederivable therein.

C. HELPER LIPIDS

In some aspects of the present disclosure, one or more lipids are mixedwith the amino lipids of the instant disclosure to create a nanoparticlecomposition. In some embodiments, the amino lipids are mixed with 1, 2,3, 4, or 5 different types of lipids. It is contemplated that the aminolipids can be mixed with multiple different lipids of a single type. Insome embodiments, the lipid could be a steroid or a steroid derivative.In other embodiments, the lipid is a PEG lipid. In other embodiments,the lipid is a phospholipid. In other embodiments, the nanoparticlecomposition comprises a steroid or a steroid derivative, a PEG lipid, aphospholipid, or any combination thereof.

1. Steroids and Steroid Derivatives

In some aspects of the present disclosure, the amino lipids are mixedwith one or more steroid or a steroid derivative to create ananoparticle composition. In some embodiments, the steroid or steroidderivative comprises any steroid or steroid derivative. As used herein,in some embodiments, the term “steroid” is a class of compounds with afour ring 17 carbon cyclic structure which can further comprises one ormore substitutions including alkyl groups, alkoxy groups, hydroxygroups, oxo groups, acyl groups, or a double bond between two or morecarbon atoms. In one aspect, the ring structure of a steroid comprisesthree fused cyclohexyl rings and a fused cyclopentyl ring as shown inthe formula below:

In some embodiments, a steroid derivative comprises the ring structureabove with one or more non-alkyl substitutions. In some embodiments, thesteroid or steroid derivative is a sterol wherein the formula is furtherdefined as:

In some embodiments of the present disclosure, the steroid or steroidderivative is a cholestane or cholestane derivative. In a cholestane,the ring structure is further defined by the formula:

As described above, a cholestane derivative includes one or morenon-alkyl substitution of the above ring system. In some embodiments,the cholestane or cholestane derivative is a cholestene or cholestenederivative or a sterol or a sterol derivative. In other embodiments, thecholestane or cholestane derivative is both a cholestere and a sterol ora derivative thereof.

In some embodiments, the present composition comprises a ratio of thecompound or amino lipids to the steroid or steroid derivative from about1:3 to about 30:1 or from about 1:1 to about 20:1. The ratio may be fromabout 1:1-6:1 such as a ratio of about 1.3:1. In some embodiments, theratio is from about 1:3, 1:2, 1:1, 1.25:1, 1.5:1, 2:1, 3:1, 5:1, 8:1,10:1, 12.5:1, 15:1, 17.5:1, 20:1, 25:1, to about 30:1, or any rangederivable therein.

2. PEG or PEGylated Lipid

In some aspects of the present disclosure, the amino lipids (orcompounds) are mixed with one or more PEGylated lipids (or PEG lipid) tocreate a nanoparticle composition. In some embodiments, the presentdisclosure comprises using any lipid to which a PEG group has beenattached. In some embodiments, the PEG lipid is a diglyceride which alsocomprises a PEG chain attached to the glycerol group. In otherembodiments, the PEG lipid is a compound which contains one or moreC6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid groupattached to a linker group with a PEG chain Some non-limiting examplesof a PEG lipid includes a PEG modified phosphatidylethanolamine andphosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylaminesand PEG modified 1,2-diacyloxypropan-3-amines, PEG modifieddiacylglycerols and dialkylglycerols. In some embodiments, PEG modifieddiastearoylphosphatidylethanolamine or PEG modifieddimyristoyl-sn-glycerol. In some embodiments, the PEG modification ismeasured by the molecular weight of PEG component of the lipid. In someembodiments, the PEG modification has a molecular weight from about 100to about 5,000. In some embodiments, the molecular weight is from about200 to about 500 or from about 1,200 to about 3,000. Some non-limitingexamples of lipids that may be used in the present disclosure are taughtby U.S. Pat. No. 5,820,873, WO 2010/141069, or U.S. Pat. No. 8,450,298,which is incorporated herein by reference.

In another aspect, the PEG lipid has the formula:

wherein: n₁ is an integer between 1 and 100 and n₂ and n₃ are eachindependently selected from an integer between 1 and 29. In someembodiments, n₁ is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100, or any range derivable therein. In someembodiments, n₁ is from about 30 to about 50. In some embodiments, n₂ isfrom 5 to 23. In some embodiments, n₂ is 11 to about 17. In someembodiments, n₃ is from 5 to 23. In some embodiments, n₃ is 11 to about17.

In some embodiments, the present composition comprises a ratio of thecompound or amino lipids to the PEG lipid from about 1:1 to about 150:1or from about 2.5:1 to about 100:1. The ratio may be from about7.5:1-50:1 such as a ratio of about 33.3:1. In some embodiments, theratio is from about 5:1, 10:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 60:1,70:1, 80:1, 90:1, 100:1, 120:1, 140:1, to about 150:1, or any rangederivable therein.

3. Phospholipid

In some aspects of the present disclosure, the amino lipids are mixedwith one or more phospholipids to create a nanoparticle composition. Insome embodiments, any lipid which also comprises a phosphate group. Insome embodiments, the phospholipid is a structure which contains one ortwo long chain C6-C24 alkyl or alkenyl groups, a glycerol or asphingosine, one or two phosphate groups, and, optionally, a smallorganic molecule. In some embodiments, the small organic molecule is anamino acid, a sugar, or an amino substituted alkoxy group, such ascholine or ethanolamine. In some embodiments, the phospholipid is aphosphatidylcholine. In some embodiments, the phospholipid isdistearoylphosphatidylcholine.

In some embodiments, the present composition comprises a ratio of thecompound or amino lipids to the phospholipid from about 1:1 to about15:1 or from about 1:1 to about 9:1. The ratio may be from about2.5:1-7.5:1 such as a ratio of about 5:1. In some embodiments, the ratiois from about 1:1, 2:1, 3:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1,10:1, 12:1, 14:1, to about 15:1, or any range derivable therein.

D. NUCLEIC ACIDS AND NUCLEIC ACID BASED THERAPEUTIC AGENTS

1. Nucleic Acids

In some aspects of the present disclosure, the nanoparticle compositionscomprise one or more nucleic acids. In addition, it should be clear thatthe present disclosure is not limited to the specific nucleic acidsdisclosed herein. The present disclosure is not limited in scope to anyparticular source, sequence, or type of nucleic acid, however, as one ofordinary skill in the art could readily identify related homologs invarious other sources of the nucleic acid including nucleic acids fromnon-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp,ape, baboon, cow, pig, horse, sheep, cat and other species). It iscontemplated that the nucleic acid used in the present disclosure cancomprises a sequence based upon a naturally-occurring sequence. Allowingfor the degeneracy of the genetic code, sequences that have at leastabout 50%, usually at least about 60%, more usually about 70%, mostusually about 80%, preferably at least about 90% and most preferablyabout 95% of nucleotides that are identical to the nucleotide sequenceof the naturally-occurring sequence. In another embodiment, the nucleicacid is a complementary sequence to a naturally occurring sequence, orcomplementary to 75%, 80%, 85%, 90%, 95% and 100%.

In some aspects, the nucleic acid is a sequence which silences, iscomplimentary to, or replaces another sequence present in vivo.Sequences of 17 bases in length should occur only once in the humangenome and, therefore, suffice to specify a unique target sequence.Although shorter oligomers are easier to make and increase in vivoaccessibility, numerous other factors are involved in determining thespecificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 orlonger are contemplated as well.

The nucleic acid used herein may be derived from genomic DNA, i.e.,cloned directly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also contemplated is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present disclosure may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

In some embodiments, the nucleic acid comprises one or more antisensesegments which inhibits expression of a gene or gene product. Antisensemethodology takes advantage of the fact that nucleic acids tend to pairwith “complementary” sequences. By complementary, it is meant thatpolynucleotides are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. That is, the largerpurines will base pair with the smaller pyrimidines to form combinationsof guanine paired with cytosine (G:C) and adenine paired with eitherthymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) inthe case of RNA. Inclusion of less common bases such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to form a siRNA or to generate specific constructs.For example, where an intron is desired in the ultimate construct, agenomic clone will need to be used. The cDNA, siRNA, or a synthesizedpolynucleotide may provide more convenient restriction sites for theremaining portion of the construct and, therefore, would be used for therest of the sequence. Other embodiments include dsRNA or ssRNA, whichmay be used to target genomic sequences or coding/non-codingtranscripts.

In other embodiments, the nanoparticles may comprise a nucleic acidwhich comprises one or more expression vectors are used in a genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Sambrook et al. (1989) and Ausubel etal. (1994), both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

2. siRNA

As mentioned above, the present disclosure contemplates the use of oneor more inhibitory nucleic acid for reducing expression and/oractivation of a gene or gene product. Examples of an inhibitory nucleicacid include but are not limited to molecules targeted to an nucleicacid sequence, such as an siRNA (small interfering RNA), short hairpinRNA (shRNA), double-stranded RNA, an antisense oligonucleotide, aribozyme and molecules targeted to a gene or gene product such as anaptamer.

An inhibitory nucleic acid may inhibit the transcription of a gene orprevent the translation of the gene transcript in a cell. An inhibitorynucleic acid may be from 16 to 1000 nucleotides long, and in certainembodiments from 18 to 100 nucleotides long.

Inhibitory nucleic acids are well known in the art. For example, siRNA,shRNA and double-stranded RNA have been described in U.S. Pat. Nos.6,506,559 and 6,573,099, as well as in U.S. Patent Publications2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161,and 2004/0064842, all of which are herein incorporated by reference intheir entirety.

Since the discovery of RNAi by Fire and colleagues in 1998, thebiochemical mechanisms have been rapidly characterized. Double strandedRNA (dsRNA) is cleaved by Dicer, which is an RNAase III familyribonuclease. This process yields siRNAs of 21 nucleotides in length.These siRNAs are incorporated into a multiprotein RNA-induced silencingcomplex (RISC) that is guided to target mRNA. RISC cleaves the targetmRNA in the middle of the complementary region. In mammalian cells, therelated microRNAs (miRNAs) are found that are short RNA fragments (˜22nucleotides). miRNAs are generated after Dicer-mediated cleavage oflonger (˜70 nucleotide) precursors with imperfect hairpin RNAstructures. The miRNA is incorporated into a miRNA-protein complex(miRNP), which leads to translational repression of target mRNA.

In designing a nucleic acid capable of generating an RNAi effect, thereare several factors that need to be considered such as the nature of thesiRNA, the durability of the silencing effect, and the choice ofdelivery system. To produce an RNAi effect, the siRNA that is introducedinto the organism will typically contain exonic sequences. Furthermore,the RNAi process is homology dependent, so the sequences must becarefully selected so as to maximize gene specificity, while minimizingthe possibility of cross-interference between homologous, but notgene-specific sequences. Particularly the siRNA exhibits greater than80, 85, 90, 95, 98% or even 100% identity between the sequence of thesiRNA and a portion of a EphA nucleotide sequence. Sequences less thanabout 80% identical to the target gene are substantially less effective.Thus, the greater identity between the siRNA and the gene to beinhibited, the less likely expression of unrelated genes will beaffected.

In addition, the size of the siRNA is an important consideration. Insome embodiments, the present disclosure relates to siRNA molecules thatinclude at least about 19-25 nucleotides, and are able to modulate geneexpression. In the context of the present disclosure, the siRNA isparticularly less than 500, 200, 100, 50, 25, or 20 nucleotides inlength. In some embodiments, the siRNA is from about 25 nucleotides toabout 35 nucleotides or from about 19 nucleotides to about 25nucleotides in length.

To improve the effectiveness of siRNA-mediated gene silencing,guidelines for selection of target sites on mRNA have been developed foroptimal design of siRNA (Soutschek et al., 2004; Wadhwa et al., 2004).These strategies may allow for rational approaches for selecting siRNAsequences to achieve maximal gene knockdown. To facilitate the entry ofsiRNA into cells and tissues, a variety of vectors including plasmidsand viral vectors such as adenovirus, lentivirus, and retrovirus havebeen used (Wadhwa et al., 2004).

Within an inhibitory nucleic acid, the components of a nucleic acid neednot be of the same type or homogenous throughout (e.g., an inhibitorynucleic acid may comprise a nucleotide and a nucleic acid or nucleotideanalog). Typically, an inhibitory nucleic acid form a double-strandedstructure; the double-stranded structure may result from two separatenucleic acids that are partially or completely complementary. In certainembodiments of the present disclosure, the inhibitory nucleic acid maycomprise only a single nucleic acid (polynucleotide) or nucleic acidanalog and form a double-stranded structure by complementing with itself(e.g., forming a hairpin loop). The double-stranded structure of theinhibitory nucleic acid may comprise 16-500 or more contiguousnucleobases, including all ranges derivable thereof. The inhibitorynucleic acid may comprise 17 to 35 contiguous nucleobases, moreparticularly 18 to 30 contiguous nucleobases, more particularly 19 to 25nucleobases, more particularly 20 to 23 contiguous nucleobases, or 20 to22 contiguous nucleobases, or 21 contiguous nucleobases that hybridizewith a complementary nucleic acid (which may be another part of the samenucleic acid or a separate complementary nucleic acid) to form adouble-stranded structure.

siRNA can be obtained from commercial sources, natural sources, or canbe synthesized using any of a number of techniques well-known to thoseof ordinary skill in the art. For example, commercial sources ofpredesigned siRNA include Invitrogen's Stealth™ Select technology(Carlsbad, Calif.), Ambion®(Austin, Tex.), and Qiagen® (Valencia,Calif.). An inhibitory nucleic acid that can be applied in thecompositions and methods of the present disclosure may be any nucleicacid sequence that has been found by any source to be a validateddownregulator of the gene or gene product.

In some embodiments, the disclosure features an isolated siRNA moleculeof at least 19 nucleotides, having at least one strand that issubstantially complementary to at least ten but no more than thirtyconsecutive nucleotides of a nucleic acid that encodes a gene, and thatreduces the expression of a gene or gene product. In one embodiments ofthe present disclosure, the siRNA molecule has at least one strand thatis substantially complementary to at least ten but no more than thirtyconsecutive nucleotides of the mRNA that encodes a gene or a geneproduct.

In one embodiments, the siRNA molecule is at least 75, 80, 85, or 90%homologous, particularly at least 95%, 99%, or 100% similar oridentical, or any percentages in between the foregoing (e.g., thedisclosure contemplates 75% and greater, 80% and greater, 85% andgreater, and so on, and said ranges are intended to include all wholenumbers in between), to at least 10 contiguous nucleotides of any of thenucleic acid sequences encoding a target therapeutic protein.

The siRNA may also comprise an alteration of one or more nucleotides.Such alterations can include the addition of non-nucleotide material,such as to the end(s) of the 19 to 25 nucleotide RNA or internally (atone or more nucleotides of the RNA). In certain aspects, the RNAmolecule contains a 3′-hydroxyl group. Nucleotides in the RNA moleculesof the present disclosure can also comprise non-standard nucleotides,including non-naturally occurring nucleotides or deoxyribonucleotides.The double-stranded oligonucleotide may contain a modified backbone, forexample, phosphorothioate, phosphorodithioate, or other modifiedbackbones known in the art, or may contain non-natural internucleosidelinkages. Additional modifications of siRNAs (e.g., 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, 5-C-methyl nucleotides, one or more phosphorothioateinternucleotide linkages, and inverted deoxyabasic residueincorporation) can be found in U.S. Publication 2004/0019001 and U.S.Pat. No. 6,673,611 (each of which is incorporated by reference in itsentirety). Collectively, all such altered nucleic acids or RNAsdescribed above are referred to as modified siRNAs.

In one embodiment, siRNA is capable of decreasing the expression of aparticular genetic product by at least 10%, at least 20%, at least 30%,or at least 40%, at least 50%, at least 60%, or at least 70%, at least75%, at least 80%, at least 90%, at least 95% or more or any ranges inbetween the foregoing.

3. tRNA

In some aspects, the present composition comprises a transfer RNA (knownas a tRNA). As used herein, the term transfer RNA or tRNA refers to bothtraditional tRNA molecules as well as tRNA molecules with one or moremodifications unless specifically noted otherwise. Transfer RNA is anRNA polymer that is about 70 to 100 nucleotides in length. Duringprotein synthesis, a tRNA delivers an amino acid to the ribosome foraddition to the growing peptide chain Active tRNAs have a 3′ CCA tailthat may be transcribed into the tRNA during its synthesis or may beadded later during post-transcriptional processing. The amino acid iscovalently attached to the 2′ or 3′ hydroxyl group of the 3′-terminalribose to form an aminoacyl-tRNA (aa-tRNA); an amino acid canspontaneously migrate from the 2′-OH to the 3′-OH and vice versa, but itis incorporated into a growing protein chain at the ribosome from the3′-OH position. A loop at the other end of the folded aa-tRNA moleculecontains a sequence of three bases known as the anticodon. When thisanticodon sequence base-pairs with a three-base codon sequence in aribosome-bound messenger RNA (mRNA), the aa-tRNA binds to the ribosomeand its amino acid is incorporated into the nascent protein chain. Sinceall tRNAs that base-pair with a specific codon are aminoacylated with asingle specific amino acid, the translation of the genetic code iseffected by tRNAs: each of the 61 non-termination codons in an mRNAdirects the binding of its cognate aa-tRNA and the addition of a singlespecific amino acid to the growing protein polymer. In some embodiments,the tRNA may comprise a mutation in the anticodon region of the tRNAsuch that the aa-tRNA base-pairs with a different codon on the mRNA. Incertain embodiments, the mutated tRNA introduces a different amino acidinto the growing protein chain than the amino acid encoded by the mRNA.In other embodiments, the mutated tRNA base-pairs with a stop codon andintroduces an amino acid instead of terminating protein synthesis,thereby allowing the nascent peptide to continue to grow. In someembodiments, a tRNA, wild-type or mutated, may read through a stop codonand introduce an amino acid instead of terminating protein synthesis. Insome embodiments, the tRNA may comprise a full-length tRNA with the3′-terminal-CCA nucleotides included. In other embodiments, tRNAslacking the 3′-terminal -A, —CA, or —CCA are made full-length in vivo bythe CCA-adding enzyme.

In other aspects, the present compositions may further comprise one ormore modified tRNA molecules including: acylated tRNA; alkylated tRNA; atRNA containing one or more bases other than adenine, cytosine, guanine,or uracil; a tRNA covalently modified by the attachment of afluorescent, affinity, reactive, spectral, or other probe moiety; a tRNAcontaining one or more ribose moieties that are methylated or otherwisemodified; aa-tRNAs that are aminoacylated with an amino acid other thanthe 20 natural amino acids, including non-natural amino acids thatfunction as a carrier for reagents or as a fluorescent, reactive,affinity, spectral, or other probe; or any combination of thesecompositions. Some examples of modified tRNA molecules are taught bySoll, et al., 1995; El Yacoubi, et al., 2012; Grosjean and Benne, etal., 1998; Hendrickson, et al., 2004; Ibba and Soll, 2000; Johnson, etal., 1995; Johnson, et al., 1982; Crowley, et al., 1994; Beier andGrimm, 2001; Torres, et al., 2014; and Björk, et al., 1987, all of whichare incorporated herein by reference.

4. mRNA

In some aspects, the present compounds and compositions may be used inthe delivery of an mRNA to a cell. Messenger RNA or mRNA are short RNAstrands which transfer the genetic code from the DNA to the ribosomes soit may be translated into a functional protein or peptide. The mRNA'sdescribed herein may be unprocessed or have undergone processing to adda poly(A) tail, be edited in vivo, or have a 5′ cap added. The presentcompositions are contemplated in the delivery of a variety of differentmRNA including those which have not undergone processing or have beenfurther processed. Additionally, these nucleic acids may be usedtherapeutically, used to produce an antibody in vivo, or in a vaccineformulation.

5. CRISPR Related RNAs

In some aspects, the present compound and compositions may be used todeliver nucleic acid sequences for use in CRISPR gene editing. TheCRISPR/Cas nuclease or CRISPR/Cas nuclease systems that may be usedherein can include a non-coding RNA molecule (guide) RNA (sgRNA), whichsequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), withnuclease functionality (e.g., two nuclease domains).

In some aspects, a Cas nuclease and sgRNA (including a fusion of crRNAspecific for the target sequence and fixed tracrRNA) are introduced intothe cell. In general, target sites at the 5′ end of the sgRNA target theCas nuclease to the target site, e.g., the gene, using complementarybase pairing. The target site may be selected based on its locationimmediately 5′ of a protospacer adjacent motif (PAM) sequence, such astypically NGG, or NAG. In this respect, the sgRNA is targeted to thedesired sequence by modifying the first 20 nucleotides of the guide RNAto correspond to the target DNA sequence. In general, a CRISPR system ischaracterized by elements that promote the formation of a CRISPR complexat the site of a target sequence. Typically, “target sequence” generallyrefers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between the target sequence and aguide sequence promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. In general, a guide sequence is any polynucleotidesequence having sufficient complementarity with a target polynucleotidesequence to hybridize with the target sequence and directsequence-specific binding of the CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between a guidesequence and its corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g. the Burrows WheelerAligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies,ELAND (Illumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, it is contemplated that the compositions describedherein may be used to delivery to one or more cells the CRISPR nucleicacids and the nuclease or may be used to direct the delivery of only thenucleic acid.

6. Modified Nucleobases

In some embodiments, the nucleic acids of the present disclosurecomprise one or more modified nucleosides comprising a modified sugarmoiety. Such compounds comprising one or more sugar-modified nucleosidesmay have desirable properties, such as enhanced nuclease stability orincreased binding affinity with a target nucleic acid relative to anoligonucleotide comprising only nucleosides comprising naturallyoccurring sugar moieties. In some embodiments, modified sugar moietiesare substituted sugar moieties. In some embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In some embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C1-C10 alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In some embodiments,substituted sugars comprise more than one non-bridging sugarsubstituent, for example, T-F-5′-methyl sugar moieties (see, e.g., PCTInternational Application WO 2008/101157, for additional 5′,2′-bissubstituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In some embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In some embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂'O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. In somesuch embodiments, the bicyclic sugar moiety comprises a bridge betweenthe 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugarsubstituents, include, but are not limited to: —[C(R_(a))(R_(b))]_(n)—,—[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or,—C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g.,WO2008/150729); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, publishedSep. 2, 2004); 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each Ris, independently, H, a protecting group, or C₁-C₁₂ alkyl;4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group(see, U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, PCT InternationalApplication WO 2008/154401).

In some embodiments, such 4′ to 2′ bridges independently comprise from 1to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein:

-   -   x is 0, 1, or 2;    -   n is 1, 2, 3, or 4;    -   each R_(a) and R_(b) is, independently, H, a protecting group,        hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂        alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted        C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,        heterocycle radical, substituted heterocycle radical,        heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,        substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,        N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl        (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and    -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted        C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂        alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted        C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle        radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,        substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH2-N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,(J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA, and (K)Methoxy(ethyleneoxy) (4′-CH(CH₂OMe)-O-2′) BNA (also referred to asconstrained MOE or cMOE).

Additional bicyclic sugar moieties are known in the art, for exampleSingh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,5561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US 2004/0171570, US2007/0287831, and US 2008/0039618; U.S. Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In some embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the .alpha.-L configuration or in the.beta.-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2)bicyclic nucleosides have been incorporated into antisenseoligonucleotides that showed antisense activity (Frieden et al., NucleicAcids Research, 2003, 21, 6365-6372).

In some embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars; PCTInternational Application WO 2007/134181, wherein LNA is substitutedwith, for example, a 5′-methyl or a 5′-vinyl group).

In some embodiments, modified sugar moieties are sugar surrogates. Insome such embodiments, the oxygen atom of the naturally occurring sugaris substituted, e.g., with a sulfer, carbon or nitrogen atom. In somesuch embodiments, such modified sugar moiety also comprises bridgingand/or non-bridging substituents as described above. For example,certain sugar surrogates comprise a 4′-sulfur atom and a substitution atthe 2′-position (see, e.g., published U.S. Patent Application US2005/0130923) and/or the 5′ position. By way of additional example,carbocyclic bicyclic nucleosides having a 4′-2′ bridge have beendescribed (see, e.g., Freier et al., Nucleic Acids Research, 1997,25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71,7731-7740).

In some embodiments, sugar surrogates comprise rings having other than5-atoms. For example, in some embodiments, a sugar surrogate comprises asix-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoro HNA(F-HNA).

In some embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In some embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ ismethyl. In some embodiments, THP nucleosides of Formula VII are providedwherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro andR₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 for other disclosed 5′,2′-bis substitutednucleosides) and replacement of the ribosyl ring oxygen atom with S andfurther substitution at the 2′-position (see U.S. Patent Publication US2005/0130923) or alternatively 5′-substitution of a bicyclic nucleicacid (see PCT International Application WO 2007/134181 wherein a4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′position with a 5′-methyl or a 5′-vinyl group). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described (see,e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In some embodiments, the present disclosure provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In some embodiments,oligonucleotides comprise one or more RNA-like nucleosides. In someembodiments, oligonucleotides comprise one or more DNA-like nucleotides.

In some embodiments, nucleosides of the present disclosure comprise oneor more unmodified nucleobases. In certain embodiments, nucleosides ofthe present disclosure comprise one or more modified nucleobases.

In some embodiments, modified nucleobases are selected from: universalbases, hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH₃) uracil and cytosine and other alkynyl derivatives of pyrimidinebases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine,3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases as definedherein. Further modified nucleobases include tricyclic pyrimidines suchas phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one),carbazole cytidine (²H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States Patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of whichis herein incorporated by reference in its entirety.

In some embodiments, the present disclosure provides oligonucleotidescomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In some embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)'O-5), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the ligand conjugatedoligonucleotides of the present disclosure involves chemically linkingto the oligonucleotide one or more additional non-ligand moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

E. KITS

The present disclosure also provides kits. Any of the componentsdisclosed herein may be combined in the form of a kit. In someembodiments, the kits comprise a polyester polymer or a composition asdescribed above or in the claims.

The kits will generally include at least one vial, test tube, flask,bottle, syringe or other container, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional containers into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in a container. In some embodiments, all ofthe nucleic acid delivery components are combined in a single container.In other embodiments, some or all of the nucleic acid deliverycomponents with the instant compounds or compositions are provided inseparate containers.

The kits of the present disclosure also will typically include packagingfor containing the various containers in close confinement forcommercial sale. Such packaging may include cardboard or injection orblow molded plastic packaging into which the desired containers areretained. A kit may also include instructions for employing the kitcomponents. Instructions may include variations that can be implemented.

F. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1 Materials and Instrumentation

Cell Culture: Calu-6 and Calu-3 cells were obtained from the AmericanType Culture Collection and cultured in RMP1 1640 (Corning) medium withL-Glutamine and 25 mM HEPES supplemented with 5% FBS (GeminiBio-Products). IB3-1 cells were kindly provided by Harvey Pollard andcultured in serum free LHC-8 (Invitrogen) medium. HEK 293 cells wereobtained from the American Type Culture Collection and cultured in DMEM(Invitrogen) supplemented with 10% FBS (Gemini Bio-Products). HeLa-Luccells and A549-Luc cells were cultured in phenol red free DMEM highglucose medium (Hyclone) supplemented with 5% FBS (Sigma-Aldrich).IGROV1 cells were cultured in RPMI 1640 (Sigma-Aldrich) with sodiumbicarbonate and L-glutamine, supplemented with 5% FBS.

Antibodies and Reagents: p53 DO-1(#sc-126) and GFP C2 (#sc-390394)antibodies were purchased from Santa Cruz Biotechnology, Inc. Actinantibody (MAB1501) was purchased from EMD Millipore. CFTR 596 antibodywas purchased from the UNC Antibody Distribution Program. Unacylated E.coli tRNA^(Phe)-Fl⁸ (P05) was purchased from tRNA Probes, Inc. RNAiMaxand Lipofectamine 2000 were purchased from Invitrogen and used followingthe supplier's recommended protocols. G418 (sc-29065) was purchased fromSanta Cruz. PTC124 (S6003) and VX-770 (S1144) were purchased fromSelleck Chemicals. 3-Isobutyl-1-methylxanthine (IBMX) (15879) andForskolin (F3917) were purchased from Sigma-Aldrich. CFTR-Inh172 wasobtained from CFFT (Cystic Fibrosis Foundation Therapeutics, Inc)

Plasmids and Site-Directed Mutagenesis of CFTR: An expression plasmid offull-length, wild-type CFTR (pBI-CFTR) was purchased from Clontech andwas mutagenized using standard protocols for site-directed mutagenesis(Sambrook et al., 1989). Site-directed mutagenesis was performed by PCRtechniques using PfuUltra High-Fidelity DNA Polymerase (Stratagene,Santa Clara, Calif.). All mutations were confirmed by DNA sequencing.Sup-tRNA^(Arg) was a gift from Carla Oliveira (Institute of MolecularPathology and Immunology of the University of Porto (IPATIMUP), Porto,Portugal.

Quantification Methods of Mature CFTR: HEK293 cells were seeded (9×10⁵cells) and transfected with CFTR plasmids. 2 μg of CFTR plasmid and 500ng of Sup-tRNA^(Arg) were co-transfected using 4 μl of Lipofectamine2000 in a 6-well format. G418 (200 μg) or PTC124 (40 μM) was added tothe media 24 hr post-transfection and remained for 48 hr. IB3-1 cellswere seeded and G418 (0-400 μG) or PTC124 (0-20 μM) was added 24 hrlater. After 48 hr, cells were lysed directly in 2× Sample Buffer((Tris-HCL 250 mM, pH 6.8, 20% Glycerol, 2.5% SDS, 0.1% Bromophenolblue). Cell lysate proteins were separated by electrophoresis on 7%/10%step (wt/vol) polyacrylamide gels using a Tris-glycine buffering systemand transferred to polyvinylidene fluoride immobilon membranes (EMDMillipore) Western blot analysis was performed using primary CFTRantibody (596) (University of North Carolina School of Medicine, ChapelHill, N.C.), actin antibody (EMD Millipore), and secondary antibodyIRdye-680RD (Li-Cor) and imaged/quantified using a Li-Cor Odyssey CLx(Li-Cor). Data was plotted using Prism 6 (Graphpad).

CFTR-Dependent Whole-Cell Current in HEK293 Cells: HEK293 cells weretransfected with the plasmids used for the CFTR maturation experiments.2 μg of CFTR plasmid and 500 ng of Sup-tRNA^(Arg) were cotransfectedusing 4 μl of Lipofectamine 2000 in a 6-well format. 24 hrpost-transfection, the whole-cell configuration of the patch-clamptechnique was used to measure the Cl-current. The pipette solutioncontained 145 mM NMDG+−Cl—, 1 mM MgCl2, 2 mM EGTA, 5 mM ATP, and 10 mMHEPES (pH 7.3 with Tris). The bath solution was 145 mM NMDG+−Cl—, 1 mMMgCl₂, 1 mM CaCl₂, 10 mM HEPES and 10 mM glucose (pH 7.4 with Tris). Thecurrent was recorded with an Axopatch 200B patch-clamp amplifier anddigitized at 2 kHz. The membrane conductance was probed by stepping themembrane potential from a holding potential of 0 mV to membranepotentials −40 and +40 mV steps for 200 ms. Whole-cell current responseswere measured in response to 10 μM forskolin plus 100 μM IBMX and 10 μMCFTRInh-172 (Inh-172). Pipettes had resistances between 3 and 5 MΩ whenfilled with pipette solution and seal resistance exceeded 8 GU. Currentrecording and analysis was performed with pClamp 9.2 software andanalyzed with Origin 8 software.

In vitro ZAL nanoparticle formulations: Lipid nanoparticles wereprepared by the ethanol dilution method. The RNA (whether an siRNA,tRNA, sgRNA, or mRNA) was diluted in acidic aqueous buffer (unlessotherwise indicated, 10 mM citric acid/sodium citrate buffer pH 3). Thelipid mix was prepared in ethanol, with the appropriate molar ratios ofZAL, cholesterol, PEG-lipid, DSPC, and or DOPE from ethanol stocksolutions of each component. Via pipette, the lipid dilution was addedto the RNA dilution at a final volumetric ratio of 1:3, rapidly mixed bypipette, and incubated for 15-20 minutes. After this incubation period,the particles were either diluted 3-fold in, or dialyzed against 1×Dulbecco's Modified PBS without calcium and magnesium (Sigma-Aldrich).Dialyses were performed in Pur-A-Lyzer Midi dialysis chambers(Sigma-Aldrich) for 1 hour per 200 μL sample per chamber.

ZAL siRNA delivery library screen: The library of ZALs functionalizedwith epoxide and acrylate hydrophobic tails was screened for siRNAdelivery efficacy in HeLa-Luc cells. In a white opaque 96-well platetissue culture plate, HeLa cells were seeded at a density of 10×10³cells per well in 100 μL growth medium (DMEM without phenol red, 5%FBS), and allowed to attach overnight. The medium was exchanged for 200μL fresh growth medium the day of the assay. Crude ZALs products wereusing a formulation lipid mixture of 50:38.5 ZAL: cholesterol, and aZAL:siRNA such that the number of hydrophobic tails in the ZAL times theZAL:siRNA mole ratio in the formulation is ˜1000, which resulted in aweight ratio range across the library of 16:1 ZAL:siRNA for the largestZAL and 45:1 ZAL:siRNA, with an average of 29.5+/−6.3 weight ratioacross the library. ZAL NP formulations were performed in a 96-wellplate by rapid mixing of ZAL lipid mix (20 μL) and siLuc dilution (60μL, 13.33 ng/μL in 10 mM citric acid-sodium citrate buffer, pH 5) at 3:1aqueous:EtOH v:v ratio with a multichannel pipette. After a 15-20 minuteincubation period, the formulations were diluted in 12 volumes (240 μL)PBS. The nanoparticles (40 μL) were added to the HeLa-Luc cells at adose of 100 ng siRNA per well. The nanoparticles were incubated with thecells for 24 h after which time the cell viability and luciferaseexpression were evaluated with the ONE-Glo+Tox Assay cell viability andluciferase assay (Promega).

sgRNA delivery to HeLa-Luc-Cas9 cells: Select ZALs were evaluated in thedelivery of single guide RNA (sgRNA) to HeLa-Luc-Cas9 cells. In a whiteopaque 96-well plate tissue culture plate, HeLa-Luc-Cas9 cells wereseeded at a density of 5×10³ cells per well in 100 μL growth medium(DMEM without phenol red, 5% FBS), and allowed to attach overnight andthen supplemented with an additional 100 μL DMEM. ZALs-sgRNAnanoparticles were formulated using the in vitro nanoparticleformulation protocol at the indicated lipid composition and weight ratio(maintaining 50:38.5 ZAL: cholesterol mole ratio, tuningPEG-lipidadditive from 5% to 0.5%, and tuning weight ratio from 20:1 ZAL:sgRNA to5:1 ZAL:sgRNA). Luciferase deletion was evaluated using a single guideRNA designed against luciferase using the CRISPR.mit.edu algorithm,while non-targeting control sgRNA (sgScr) was used as a negativecontrol. The nanoparticles were added to the cells at a dose of 50 ngsgRNA per well and incubated with the cells for 24 h or 48 h. RNAiMax(Invitrogen) formulated according to the manufacturer's protocol withsgLuc or sgScr was used as a positive control. After 24 h or 48 h, thecell viability and luciferase expression were evaluated with theONE-Glo+Tox Assay cell viability and luciferase assay (Promega).

Co-delivery of Cas9 mRNA and sgRNA A549 and HeLa-Luc cells: ZA3 ZAL wereevaluated in the co-delivery of Cas9 mRNA (Tri-Link biotechnologies) andsingle guide RNA (sgRNA) to luciferase expressing cancer cells. In awhite opaque 96-well plate tissue culture plate, A549-Luc or HeLa-Luccells were seeded at a density of 5×10³ cells per well in 100 μL growthmedium (DMEM without phenol red, 5% FBS), and allowed to attachovernight and then supplemented with an additional 100 μL DMEM.ZALs-Cas9mRNA nanoparticles were formulated using the in vitronanoparticle formulation protocol at the indicated lipid composition andweight ratio (maintaining 50:38.5 ZAL: cholesterol mole ratio, tuningPEG-lipid additive from 5% to 0.5%, and tuning weight ratio from 20:1ZAL:sgRNA to 5:1 ZAL:sgRNA).

Different dosing reigments were evaluated including sgRNA and Cas9 mRNAin the same nanoparticle (where the sgRNA and Cas9 mRNA were diluted inthe acidic buffer dilution prior to the addition of the lipid mixture)sgRNA and Cas9 mRNA formulated in different ZAL particles but addedsimultaneously, or Cas9 mRNA added 24 h prior to the addition of sgRNA.As a negative control, sgRNA delivery in the absence of Cas9 mRNA wasalso included for all ZAL NPs tested. The nanoparticles were added tothe cells at a dose of 100 ng Cas9 mRNA or 50 ng sgRNA per well andincubated with the cells for 48 h. As a positive control Lipofectamine3000 (Invitrogen) was used to deliver Cas9 mRNA while RNAiMax(Invitrogen) formulated according to the manufacturer's protocol. 48 hafter the sgRNA deliver, the cell viability and luciferase expressionwere evaluated with the ONE-Glo+Tox Assay cell viability and luciferaseassay (Promega).

CSAL in vitro siRNA delivery efficacy: In a white opaque 96-well platetissue culture plate, HeLa-Luc or A549-Luc cells were seeded at adensity of 10×10³ cells per well in 100 μL growth medium (DMEM withoutphenol red, 5% FBS), and allowed to attach overnight. The medium wasexchanged for 200 μL fresh growth medium the day of the assay. CSALproducts were using a formulation lipid mixture of 50:38.5:10:1.5ZAL:cholesterol:DSPC:PEG-lipid, and screened at a mole ratio CSAL:siRNAof 666:1, 333:1 and 167:1. ZAL NP formulations were performed in a96-well plate by rapid mixing of CSAL lipid mix (10 μL) and siLucdilution (20 μL, 40 ng/μL in 10 mM citrate phosphate buffer, pH 3) at2:1 aqueous:EtOH v:v ratio with a multichannel pipette. After a 15-20minute incubation period, the formulations were diluted in 12 volumes(120 μL) PBS. The nanoparticles (18.75 μL) were added to the HeLa-Luccells at a dose of 100 ng siRNA per well. The nanoparticles wereincubated with the cells for 24 h after which time the cell viabilityand luciferase expression were evaluated with the ONE-Glo+Tox Assay cellviability and luciferase assay (Promega) and normalized to untreatedcells (N=3 or 4+/− standard deviation).

siRNA Uptake Studies: Cellular uptake studies were performed using CSALsNPs with the same formulation as the in vitro delivery efficacy screenin HeLa-Luc cells and A549-Luc cells. Cells were seeded at a density of30,000 cells per well in 8-chambered coverglass slides (Nunc) andallowed to attached for 24 hours. The nanoparticles were added to thecells at a final siRNA concentration of 34 nM. After 4 h or 24 hincubation, the medium was aspirated, washed with PBS, and cell membranestaining was performed (Cell Mask Green, Molecular Probes) using themanufacturer's protocol. Cells were fixed with 4% paraformaldehyde (15minutes RT), washed with PBS 2 times 5 minutes, the cell nuclei werestained with DAPI (Sigma-Aldrich) and washed with PBS. Confocalmicroscopy imaging was performed using a Zeiss LSM 700 microscope andimages were analyzed using ImageJ (NIH).

Nucleic acid binding experiments: Nucleic acid binding was evaluatedusing the Ribogreen assay (Molecular Probes). In short, nanoparticleswere prepared using the in vitro or in vivo formulation protocols. Thenanoparticle formulations (5 μL) were added to a black 96-well opaquemicroplate (Corning). A standard curve of the appropriate nucleic acidwas prepared in the same medium as the nanoparticles. Ribogreen reagentwas diluted 1:1000 in 1× PBS and 50 μL was added to each well viamultichannel pipette. The mixture was stirred on an orbital mixer for 10minutes, and the fluorescence of each well was read using a plate reader(λ_(Ex) 485 nm, λ_(Em) 535 nm). The amount of free nucleic acid wasdetermined by fitting the signal from each nanoparticle sample to thenucleic acid standard curve, and the fraction bound determined by thefollowing formula: Fraction nucleic acid bound=(total nucleic acidinput-free nucleic acid)/total nucleic acid input) (N=3 or 4+/− standarddeviation).

ZAL mRNA delivery in vitro assay: ZAL nanoparticles with fireflyluciferase mRNA (Tri-Link Biotechnologies) were prepared using the invitro nanoparticle formulation method outlined above. IGROV1 cells wereseed in white opaque 96-well tissue culture plates at a seeding densityof 5×10³ cells per well in 100 μL RPMI 1640 medium supplemented with 5%FBS, and allowed to attach overnight. After overnight incubation, andadditional 100 μL medium was added to the wells. The ZAL:mRNAnanoparticles were prepared at a ZAL:mRNA weight ratio of 20:1, 10:1,7.5:1 and 5:1, and lipid mixture molar compositions of 50:38.5ZAL:cholesterol, with PEG lipid supplemented at a molar ratio of 5%, 2%,1% or 0.5% at each weight ratio. The ZAL-mRNA nanoparticles were addedto the cells at a dose of 100 ng mRNA per well and incubated for theindicated time (ranging from 6 h to 48 h), after which time cellviability and luciferase expression were evaluated with the ONE-Glo+ToxAssay cell viability and luciferase assay (Promega) and normalized tountreated cells (N=4+/− standard deviation)

In vivo nanoparticle formulations: In vivo nanoparticle formulationswere performed using the NanoAssemblr microfluidic mixing system(Precision Nanosystems). Lipids were dissolved in ethanol and nucleicacids (mRNA or siRNA) were diluted in 10 mM citric acid-sodium citratebuffer pH 3. The lipid mixture and nucleic acid dilution were combinedat a volumetric ratio of 3:1 nucleic acid: lipid mix at a total flowrate of 12 mL per minute, and a waste collection of 0.1 mL in thebeginning and end of each formulation. The nanoparticles were dialyzedagainst 1× PBS in Pur-A-Lyzer midi dialysis chambers (Sigma-Aldrich) for1 hour per 200 μL volume in each chamber, and diluted in 1× PBS to theappropriate nucleic acid concentration.

In vivo siRNA nanoparticle biodistribution: All experiments wereapproved by the Institutional Animal Care & Use Committee (IACUC) of TheUniversity of Texas Southwestern Medical Center and were consistent withlocal, state and federal regulations as applicable. CSAL nanoparticleswere prepared using the in vivo nanoparticle formulation method at alipid mixture mole ratio of 50:38.5:10:1.5 CSAL: cholesterol: DSPC:PEG-lipid, and weight ratio ranging from 20:1 to 45:1 total lipid:siRNAweight ratio. For the siRNA dilution, the siRNA was spiked with 50%Cy5.5 labeled siRNA, and formulation performed as normal. Afterdialysis, the nanoparticles were diluted to a concentration of 1 μg per10 μL formulation. This formulation was injected at a dose of 1 mg/kgsiRNA by tail vein injection into Black 6 mice. After 2 h or 24 h time,the animals were anesthetized under isofluorane, sacrificed by cervicaldislocation, and the organs resected. Fluorescence imaging of the organswas performed on an IVIS Lumina system (PerkinELmer) using the Cy5excitation and emission filter set, and the images processed usingLiving Image analysis software (PerkinElmer).

In vivo luciferase mRNA delivery: All experiments were approved by theInstitutional Animal Care & Use Committee (IACUC) of The University ofTexas Southwestern Medical Center and were consistent with local, stateand federal regulations as applicable. ZA3-Ep10 ZAL was formulated within vivo formulation at 50 ZAL:38.5 cholesterol: 2 or 0.5 PEG-lipid moleratio in the lipid mix, and 7.5:1 ZAL:mRNA weight ratio. AthymicNude-Foxn1^(nu) mice (Harlan Laboratories) were injected with ZAL-mRNANPs at a dose of 1 mg/kg via tail vein injection or intraperiotnealinjection. After 24 h and 48 h the luciferase expression was evaluatedby live animal bioluminescence imaging Animals were anesthetized underisofluorane, and D-luciferin monosodium hydrate (GoldBio) substrate wasinjected IP. After 10-12 minute incubation, the luciferase activity byimaged on an IVIS Lumina system (PerkinELmer), and the images processedusing Living Image analysis software (PerkinElmer). Ex vivo imaging wasperformed on systemic organs after resection, and the tissue frozen ondry ice for ex vivo luciferase expression analysis.

In vivo luciferase silencing in A549 xenografts: All experiments wereapproved by the Institutional Animal Care & Use Committee (IACUC) of TheUniversity of Texas Southwestern Medical Center and were consistent withlocal, state and federal regulations as applicable. AthymicNude-Foxn1^(nu) mice (Harlan Laboratories) were implanted withxenografts in each hind flank with firefly luciferase expressing A549(5×10⁶ cells suspended in 100 μL of 1:1 v:v PBS: Matrigel (Corning)).After the tumors reached adequate size, each tumor on the same animalwas injected with in vivo formulated NPs (˜50 μL per tumor) of CSALA3OAcC2Me, with a lipid molar ratio of 50 CSAL: 38.5 cholesterol: 10DSPC: 1.5 PEG-lipid, and total lipid:siRNA weight ratio of 30:1, andfinal siRNA dose of 1 mg/kg siLuc or siCtrl. After 24 h and 48 h theluciferase expression was evaluated by live animal bioluminescenceimaging Animals were anesthetized under isofluorane, and D-luciferinmonosodium hydrate (GoldBio) substrate was injected IP. After 10-12minute incubation, the luciferase activity by imaged on an IVIS Luminasystem (PerkinELmer), and the images processed using Living Imageanalysis software (PerkinElmer).

Ex vivo luciferase expression analysis in A549 xenografts: 48 h postinjection of A3OAcC2Me siLuc or siCtrl the mice were euthanized bycervical dislocation and the A549 xenografts were resected and frozen ondry ice. The tumors were weighed on a balance, cut into strips with astraight razor and diluted at 1:3 tumor mass:volume (mg:μL) of 1×reporter lysis buffer (Promega) supplemented with protease inhibitormini tablets (Pierce) and kept on ice. The tissue was homogenized andthe luciferase expression evaluated by the Luciferase assay system.

Nanoparticle property characterization: Physical properties weremeasured using a Zetasizer Nano ZS (Malvern) with an He—Ne laser (λ=632nm). Particle sizes were measured by dynamic light scattering (BLS) (5measurements, 3 runs×10 seconds, automatic attenuator setting) by 173°back scattering. Zeta potential was measured in a folded capillary cell(Malvern) with samples diluted in PBS for ZAL NPs or citrate phosphatebuffer pH 7.4 for CSAL NPs.

tRNA Uptake Studies: Cellular uptake studies were performed using thetop performing materials from the screen. Calu6 cells were seeded at adensity of 30,000 cells per well in 8-chambered coverglass slides (Nunc)and allowed to attached for 24 hours. NP formulations were preparedusing the in vitro nanoparticle formulation procedure. The nanoparticleswere added to the cells at a final tRNA concentration of 0.9 μg/well.After 6 h incubation, the medium was aspirated, washed with PBS, andcell membrane staining was performed (Cell Mask Orange, MolecularProbes) using the manufacturer's protocol. Cells were fixed with 4%paraformaldehyde (15 minutes RT), washed with PBS 2 times 5 minutes, thecell nuclei were stained with DAPI (Sigma-Aldrich) and washed with PBS.Confocal microscopy imaging was performed using a Zeiss LSM 700microscope and images were analyzed using lmageJ (NIH).

Nanoparticle Carrier Screen in Calu6 Cells: Calu6 cells were seeded at adensity of 500,000 cells per well in a 6-well format and allowed toattach overnight. For plasmid DNA; 1 μg was transfected using 3 μl ofLipofectamine 2000 using manufacturer recommend protocols. FortRNA^(ArgOp)-RNAiMax, 4 μg was transfected using 3 μl of RNAiMax usingmanufacturer recommended protocols. Particles were diluted in Opti-MEM(Invitrogen). G418 (50 μg) and PTC124 (10 μl M) was added directly tothe media. Nanoparticles were formulated as follows. Functionalpolyester-RNA polyplexes were prepared using a weight ratio of 30:1polymer:RNA by adding 10 μL polymer stock (15 g/L in DMSO) to a dilutionof tRNA (5 μg tRNA in 490 μL 10 mM citrate buffer pH 4.2) and incubatingfor 20 minutes. Deudrimer, ZAL, and CSAL nanoparticles were preparedusing the in vitro nanoparticle formulation method detailed above.Dendrimers were formulated with a lipid mixture of 50:38:10:2dendrimer:cholesterol:DSPC:PEG-lipid, and a dendrimer:tRNA mole ratio of200:1 unless otherwise indicated. ZAL-tRNA NPs were formulated with alipid mixture of 50:38.5 ZAL:cholesterol and a total lipid:tRNA weightratio of 25:1. CSAL-tRNA NPs were formulated with a CSAL:tRNA weightratio of 20:1. For all nanoparticles, 400 μL of each formulation wasadded to the cells in 2 mL medium for a dose of 4 μg tRNA per well.After 48 hr, cells were lysed directly in 2× Sample Buffer ((Tris-HCL250 mM, pH 6.8, 20% Glycerol, 2.5% SDS, 0.1% Bromophenol blue). Celllysate proteins were separated by electrophoresis on 10% (wt/vol)polyacrylamide gels using a Tris-glycine buffering system andtransferred to polyvinylidene fluoride Immobilon membranes (EMDMillipore). Western blot analysis was performed using primary p53antibody (Santa Cruz Biotechnology, True) actin antibody (EMI)Millipore), and secondary antibody IRdye-680RD (Li-Cor) andimaged/quantified using a Li-Cor Odyssey CLx (Li-Cor).

Example 2 Synthesis and Characterization of the Amino Lipids

The cationic sulfonamide amino lipids (CSALs) were prepared usingdifferent headgroups, linker amides, with a variety of functionalsidearms for the lipid groups as shown in FIG. 1. An exemplary syntheticroute for preparing the cationic sulfonamide amino lipids is shown inFIG. 2. Some exemplary characterization information for CSAL A3OAcC2Meis shown in FIG. 40. Alternative synthesis methods are described in FIG.42.

Synthesis of A1-OAc-Cn-Me/Et and A2-OAc-Cn-Me/Et CSALs: In a 20 mL vialequipped with a stir bar was dissolved A1-OAc propanesulfonate (100 mg,0.136 mmol) or A2-OAc propanesulfonate (in 2 mL thionyl chloride. Thevial was sealed and the reaction mixture heated to 85° C. for 30minutes. The reaction was cooled to room temperature, diluted in 5 mLfreshly distilled toluene and concentrated under reduced pressure. Thecrude sulfonyl chloride intermediate was cooled on ice and to this wasadded the appropriate N,N-dimethyl diamine or N,N-diethyl diamine (5equiv) dissolved in 5 mL dry acetonitrile. The reaction mixture wasstirred on ice for 15 minutes, and the reaction mixture concentratedunder reduced pressure. The crude product was purified on silica gelwith a solvent gradient of 5% MeOH in DCM to 20% MeOH, 1% sat. NH₄OH inDCM to yield the product as a sticky yellow or brown solid.

-   A1OAcC2Me Mass calculated m/z 803.6654, observed (MALDI-TOF ms) m/z    803.3930-   A1OAcC3Me Mass calculated m/z 817.6810, observed (MALDI-TOF ms) m/z    817.5598-   A1OAcC4Me Mass calculated m/z 831.6967, observed (MALDI-TOF ms) m/z    831.5186-   A1OAcC2Et Mass calculated m/z 831.6967, observed (MALDI-TOF ms) m/z    831.80-   A1OAcC3Et Mass calculated m/z 845.7123, observed (MALDI-TOF ms) m/z    846.51-   A2OAcC2Me Mass calculated m/z 831.6967, observed M⁺¹ (MALDI-TOF ms)    m/z 832.62-   A2OAcC2Et Mass calculated m/z 859.7280, observed M⁺¹ (MALDI-TOF ms)    m/z 860.66

Synthesis of A3-OAc-C2Me: In a 20 mL vial equipped with a stir bar wasdissolved A3-OAc propanesulfonate (200 mg, 0.155 mmol) in 2 mL thionylchloride. The vial was sealed and the reaction mixture heated to 85° C.for 30 minutes. The reaction was cooled to room temperature, diluted in5 mL freshly distilled toluene and concentrated under reduced pressure.The crude sulfonyl chloride intermediate was cooled on ice and to thiswas added the appropriate N,N-dimethyl ethylenediamine (0.775 mmol, 85μL, 5 equiv) dissolved in 5 mL dry acetonitrile. The reaction mixturewas stirred on ice for 15 minutes, and the reaction mixture concentratedunder reduced pressure. The crude product was purified on silica gelwith a solvent gradient of 5% MeOH in DCM to 20% MeOH, 1% sat. NH₄OH inDCM to yield the product as a sticky brown solid (79.8 mg, 38.0% yield).Mass calculated m/z 1355.1567, observed M⁺¹ (MALDI-TOF ms) m/z 1355.18.

Synthesis of A1-OPiv-CnMe CSALs: In a 20 mL vial equipped with a stirbar was dissolved A1-OPiv propanesulfonate (100 mg, 0.122 mmol) in 2 mLthionyl chloride. The vial was sealed and the reaction mixture heated to85° C. for 30 minutes. The reaction was cooled to room temperature,diluted in 5 mL freshly distilled toluene and concentrated under reducedpressure. The crude sulfonyl chloride intermediate was cooled on ice andto this was added the appropriate N,N-dimethyl diamine (5 equiv)dissolved in 5 mL dry acetonitrile. The reaction mixture was stirred onice for 15 minutes, and the reaction mixture concentrated under reducedpressure. The crude product was purified on silica gel with a solventgradient of 5% methanol in DCM to 75% DCM, 20% methanol, 5% saturatedammonium hydroxide in water to yield the product as a sticky yellow orbrown solid.

-   A1OPivC2Me Mass calculated m/z 887.7593, observed M⁺¹ (MALDI-TOF ms)    m/z 887.7920-   A1OPivC3Me Mass calculated m/z 901.7749, observed M⁺¹ (MALDI-TOF ms)    m/z 901.4854-   A1OPivC4Me Mass calculated m/z 915.7906, observed M⁺¹ (MALDI-TOF ms)    m/z 915.6368

Synthesis of A1-Cl-CnMe CSALs: In a 20 mL vial equipped with a stir barwas dissolved A1-OH propanesulfonate (100 mg, 0.154 mmol) in 2 mLthionyl chloride. The vial was sealed and the reaction mixture heated to85° C. for 1 hour. The reaction was cooled to room temperature, dilutedin 5 mL freshly distilled toluene and concentrated under reducedpressure. The crude sulfonyl chloride intermediate was cooled on ice andto this was added the appropriate N,N-dimethyl diamine (5 equiv)dissolved in 5 mL dry acetonitrile. The reaction mixture was stirred onice for 15 minutes, and the reaction mixture concentrated under reducedpressure, and dried under vacuum.

-   A1ClC2Me Mass calculated m/z 755.5765, observed M⁺¹ (MALDI-TOF ms)    m/z 755.7258-   A1ClC3Me Mass calculated m/z 769.5921, observed M⁺¹ (MALDI-TOF ms)    m/z 769.6628-   A1ClC4Me Mass calculated m/z 783.6078, observed M⁺¹ (MALDI-TOF ms)    m/z 783.7239

Synthesis of A1OHC2Me: In a 20 mL vial equipped with a stir bar wasdissolved A1-OAc propanesulfonate (100 mg, 0.136 mmol) in 2 mL thionylchloride. The vial was sealed and the reaction mixture heated to 85° C.for 30 minutes. The reaction was cooled to room temperature, diluted in5 mL freshly distilled toluene and concentrated under reduced pressure.The crude sulfonyl chloride intermediate was cooled on ice and to thiswas added the appropriate N,N-dimethyl-ethylenediamine (85.6 μL, 0.68mmol, 5 equiv) dissolved in 5 mL dry acetonitrile. The reaction mixturewas stirred on ice for 15 minutes, and the reaction mixture concentratedunder reduced pressure. The reaction mixture was redissolved in 5 mLmethanol and potassium carbonate (0.93 g, 0.68 mmol, 5 equiv) was addedand the reaction mixture stirred at 40° C. for 4 days. After reaction,the mixture was cooled, filtered, and concentrated under reducedpressure. The concentrate was dissolved in acetone and additionalprecipitate was removed by filtration to yield the crude product as ayellow sticky solid. The product was purified over silica gel (5%methanol in DCM to 20% methanol, 2% saturated ammonium hydroxide indichloromethane to yield the product as a sticky yellow solid (17.5 mg,17.9% yield). Mass calculated m/z 719.6443, observed M⁺¹ (MALDI-TOF ms)m/z 719.8963.

Similarly, the zwitterionic amino lipids (ZALs) were prepared from thestarting components shown in FIG. 9. As shown in FIG. 10, thezwitterionic head group was synthesized and the appropriate ¹H NMRspectra for the starting material and the zwitterionic head group areshown. The zwitterionic head group was reacted with the poly amine coreto obtain the compound shown in FIG. 11 with the corresponding ¹H NMRspectra. Several of the compounds with different polyamine core and thezwitterionic head group are shown in FIG. 12. The reaction of these headgroup and cores is shown in FIG. 13 and with the appropriate reactionconditions for the three different lipid reactive groups in FIG. 14.LCMS analysis of three compounds is shown in FIGS. 15A-15C. Someexemplary characterization informations for several ZALs including FIGS.36 & 37. Alternative synthesis methods are described in FIGS. 39 & 40.

Synthesis of 3-((2-acrylamidoethyl)dimethylammonio)propane-1-sulfonate(SBAm): A flame-dried 500 mL round-bottom flask equipped with a stirbar, and an addition funnel under a nitrogen atmosphere was charged withN,N-dimethyl ethenediamine (20 g, 226.9 mmol) and triethylamine (1equiv, 227 mmol, 31.6 mL) in 250 mL dry THF, and cooled to 0° C.Acryloyl chloride (0.9 equiv, 204.2 mmol, 16.6 mL) was dissolvedseparately in 50 mL dry THF and added dropwise via the addition funnelto the stirring amine solution. The reaction was allowed to warm to roomtemperature overnight which resulted in a yellow solution with whiteprecipitate. The precipitate was filtered off and the filtrate wasconcentrated in vacuo. The crude product was purified by silica gelcolumn (20% MeOH in DCM). The product was dried with anhydrous sodiumsulfate and concentrated under reduced pressure to yield thedimethylamino acrylamide intermediate as an orange liquid (9.36 g, 32.2%yield for step 1).

In a 250 mL round-bottom flask equipped with a stir bar, thedimethylamino acrylamide intermediate (9.36 g, 65.8 mmol) was dissolvedin 100 mL acetone. In one portion, 1,3-propanesultone (1.1 equiv, 72.4mmol, 8.85 g) was added. A rubber stopper with a needle vent wasinstalled and the reaction mixture was heated to 50° C. overnight,yielding the formation of an off white solid precipitate. Theprecipitate was collected by vacuum filtration, washed with copiousamounts of acetone, and dried under vacuum overnight yielding the SBAmproduct as an light yellow solid (14.77 g, 84.9% yield for step 2). Masscalculated m/z 264.11, observed M⁺¹ (LCMS direct inject) m/z 265.1.

Amino SBAm Syntheses for Library Preparation:

General synthesis of propanesulfonate amide-bearing zwitterionic amines(Amino SBAms) In a 20 mL vial equipped with a stir bar,3-((2-acrylamidoethyl)dimethylammonio)propane-1-sulfonate (SBAm, 1.5 g,5.67 mmol, 1 equiv) was dissolved in 5.67 mL deionized water to aconcentration of 1M. The corresponding amine (28.35 mmol, 5 equiv) wasadded via pipette in one portion, the vial covered and stirred at roomtemperature overnight. After overnight reaction, the amino SBAm reactionmixture was transferred to several 50 mL polypropylene conical tubes wasprecipitated in >10 volumes acetone to remove the residual aminestarting material, collected by centrifugation (4000×g, 10 minutes). Thesupernatant was decanted, the pellet washed with acetone, and driedunder vacuum to yield the amino SBAms.

ZA1: Light yellow sticky solid (2.40 g, 93.6% yield). Mass calculatedm/z 452.31, observed M^(|1) (LCMS direct inject) m/z 453.3. ¹H NMR (400MHz, D₂O) δ 3.65 (t, J=6.8 Hz, 2H), 3.48 (ddd, J=13.7, 9.5, 5.7 Hz, 4H),3.14 (s, 6H), 2.95 (t, J=7.2 Hz, 2H), 2.68 (s, 2H), 2.65-2.54 (m, 14H),2.54-2.48 (m, 2H), 2.29 (d, J=1.0 Hz, 9H), 2.22-2.16 (m, 4H).

ZA2: Reaction done on a 0.776 g SBAm scale. Viscous yellow oil (0.36 g,24.8% yield). Mass calculated m/z 536.41, observed M⁺¹ (LCMS directinject) m/z 537.4. ¹H NMR (500 MHz, D₂O) δ 3.50 (t, J=7.0 Hz, 2H), 3.33(ddd, J=22.0, 11.2, 5.7 Hz, 4H), 2.98 (s, 6H), 2.83-2.62 (m, 4H), 2.57(dt, J=21.3, 7.3 Hz, 4H), 2.44 (p, J=7.1 Hz, 6H), 2.30-2.23 (m, 1H),2.11-2.01 (m, 2H), 0.92-0.86 (m, 9H), 0.84 (d, J=6.5 Hz, 4H).

ZA3: Brown sticky solid (2.61 g, quantitative yield). Mass calculatedm/z 410.58, observed M⁺¹ (LCMS direct inject) m/z 411.3. ¹H NMR (500MHz, D₂O) δ 3.62 (t, J=6.7 Hz, 2H), 3.50-3.40 (m, 4H), 3.11 (d, J=1.4Hz, 6H), 2.92 (td, J=7.2, 1.3 Hz, 2H), 2.82-2.68 (m, 5H), 2.66-2.49 (m,8H), 2.41 (ddd, J=8.2, 5.9, 1.3 Hz, 2H), 2.23-2.14 (m, 2H).

ZA4: Light yellow sticky solid (2.01 g, 92.9% yield) Mass calculated m/z381.24, observed M⁺¹ (LCMS direct inject) m/z 382.2. ¹H NMR (400 MHz,D₂O) δ 3.66 (t, J=6.8 Hz, 2H), 3.49 (ddd, J=13.7, 8.7, 5.8 Hz, 4H), 3.14(s, 6H), 2.96 (t, J=7.2 Hz, 2H), 2.86-2.64 (m, 6H), 2.57-2.40 (m, 5H),2.28-2.14 (m, 6H).

ZA5: Sticky yellow solid (2.32 g, 84.1% yield). Mass calculated m/z409.27, observed M⁺¹ (LCMS direct inject) m/z 410.2. ¹H NMR (400 MHz,D₂O) δ 3.52 (t, J=6.8 Hz, 3H), 3.35 (ddd, J=13.8, 9.0, 5.6 Hz, 5H), 3.00(s, 7H), 2.82 (t, J=7.2 Hz, 3H), 2.65 (t, J=7.1 Hz, 3H), 2.49 (q, J=6.4,5.5 Hz, 1H), 2.39 (t, J=7.4 Hz, 2H), 2.26 (dq, J=15.4, 5.4, 3.7 Hz, 7H),2.14-1.99 (m, 7H), 1.55-1.41 (m, 4H).

ZA6: Sticky yellow solid (2.71 g, quantitative yield). Mass calculatedm/z 464.31, observed M⁺¹ (LCMS direct inject) m/z 465.3. ¹H NMR (500MHz, D₂O) δ 3.64 (t, J=6.9 Hz, 2H), 3.52-3.42 (m, 4H), 3.12 (s, 7H),2.94 (t, J=7.2 Hz, 3H), 2.82-2.68 (m, 5H), 2.53 (t, J=7.4 Hz, 2H),2.45-2.30 (m, 7H), 2.26-2.15 (m, 4H), 1.64 (tdd, J=15.5, 12.1, 7.6 Hz,4H).

Synthesis of Amino SBAm Epoxide and Acrylate Libraries of ZwitterionicAmino Lipids (ZALs):

A zwitterionic amino lipid (ZAL) library of all previously describedamino SBAms functionalized was prepared by introduction of hydrophobictails through reaction with with 1,2-epoxy alkanes and hydrophobicacrylates. The epoxides (1,2-epoxyoctane, 1,2-epoxydecane,1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and1,2-epoxyoctadecane)were purchased commercially and encoded to includethe total number of carbon atoms in the molecule (Cn, 8-18). Thehydrophobic acrylates (were either purchased commercially (O12, O18) orsynthesized by the reaction of the appropriate primary alcohol withacryloyl chloride (O8, O10, O14, O16), and encoded to include the numberof carbon atoms in the hydrophobic tail, but not including the acrylatemoiety. To prepare the library, in a 4 mL vial equipped with a stir bar,the zwitterionic amines (0.1 mmol or 0.05 mmol) were weighed out bybalance, and dissolved to a concentration of 1 M in iPrOH for epoxideZALs or in DMSO for acrylate ZALs. The appropriate hydrophobicelectrophile was added with N equivalents, where N is the number ofamine reactive sites that would yield complete conversion of primary andsecondary amines to tertiary amines The vials were sealed and thereactions stirred for several days at 75° C. for epoxides and 80° C. foracrylates. After reaction, the reactions were precipitated in acetone toyield the zwitterionic aminolipids.

Alternative Synthesis of ZA3: A 20 mL vial equipped with a stir bar wascharged with 3-((2-acrylamidoethyl)dimethylammonio)propane-1-sulfonate(SBAm, 0.8111 g, 3.068 mmol) and dissolved in 3 mL DMSO. Via syringe,tris(2-aminoethyl) amine (5 equiv, 15.32 mmol, 2.24 g) was addedyielding a cloudy yellow/brown suspension. The reaction mixture wassealed and stirred at 80° C. overnight, yielding an orange cloudysuspension. The reaction mixture was further diluted in DMSO,transferred to several 50 mL conical tubes and precipitated in 10volumes ethyl acetate. The precipitate collected by centrifugation(4,000×g, 10 minutes), and the supernatant decanted to yield a stickyyellow/brown. The product was reprecipitated in DMSO/EtOAc several timesto remove any residual tris(2-aminoethyl) amine, and finally dissolvedin MeOH transferred to round-bottom flask and concentrated under reducedpressure. The product was dried overnight under vacuum to removeresidual solvent, redissolved in methanol and precipitated in ethylacetate, and dried under vacuum to yield ZA3 as an orange/brown oil(1.4058 g, 100%).

Synthesis of ZA3-Ep10: A 20 mL scintillation vial equipped with a stirbar was charged with ZA3 (300 mg, 0.7307 mmol) and iPrOH (730 μL, 1MSBAm) and stirred briefly at RT to yield a yellow/brown suspension.1,2-epoxydecane (4.384 mmol, 685 mg, 6 equiv) was added, the vial wassealed and stirred overnight at 75° C. for approximately 24 h resultingin a clear yellow/brown solution. The iPrOH was removed under reducedpressure to yield a yellow/brown oil. The crude product was dissolved inminimal 5% MeOH in DCM and purification was carried out on a silica gelcolumn (24 g) using the CombiFlash® system (Teledyne Isco). The productwas eluted and fractionated with a solvent gradient of 5% MeOH in DCM to20% MeOH, 2% saturated ammonium hydroxide in DCM and the product elutiontracked by ELSD. The product containing fractions were concentratedunder reduced pressure, and dried under vacuum overnight to yield theproduct as a sticky yellow solid (192.5 mg, 22.1% yield). Masscalculated m/z 1191.0246, observed M⁺¹ (LCMS direct inject) m/z 1192.8.

Synthesis of propanesulfonate A1-OH: In a 250 mL round-bottom flaskequipped with a stir bar,1,1′-(3-(dimethylamino)propyl)azanediyl)bis(tetradecan-2-ol) (6.37 g,12.09 mmol) was dissolved in 50 mL acetone, followed 1,3-propanesultone(2.21 g, 18.13 mmol, 1.5 equiv). The flask was covered and stirred at50° C. overnight, which resulted in the formation of a whiteprecipitate. The white precipitate was collected by filtration, anddried under vacuum to yield the propanesulfonate product as a whitesolid (7.46 g, 95.0%). Mass calculated m/z 648.5475, observed M⁺¹(MALDI-TOF ms) m/z 649.8078.

Synthesis of propanesulfonate A2-OH: In a 20 mL vial equipped with astir bar, A2-OH (0.5 g, 0.901 mmol) was dissolved in 4 mL acetone,followed by the addition of 1,3-propanesultone (165 mg, 1.35 mmol, 1.5equiv). The vial was sealed and stirred overnight at 50° C. Afterovernight reaction, an additional 1.5 equiv 1,3-propanesultone was addedand stirred for an additional day. The reaction mixture wasconcentrated, dissolved in minimal dichloromethane, and purified oversilica gel (gradient 10% MeOH in DCM to 10% MeOH, 1% sat. NH₄OH in DCM)to yield the product as a sticky pale yellow solid (310 mg, 50.8%yield). Mass calculated m/z 676.5788, observed M⁺¹ (MALDI-TOF ms) m/z678.22

Synthesis of propanesulfonate A3-OH: Same protocol as propanesulfonateA2-OH sticky pale yellow solid (2.864 g, 85.0% yield) Mass calculatedm/z 1116.0177, observed M⁺¹ (MALDI-TOF ms) m/z 1117.34

Synthesis of propanesulfonate A1-OAc: In a 20 mL vial equipped with astir bar, propanesulfonate A1-OH (1.25 g, 1.93 mmol) was dissolved in 5mL dichloromethane, followed by the addition acetic anhydride (10 mL,excess). The reaction mixture was stirred at room temperature for 3 daysuntil the consumption of starting material by TLC. The reaction mixturewas diluted in acetone and concentrated under reduced pressure to form aclear oil, which formed a colorless precipitate on standing. Thisprecipitate was collected by filtration, and dried under vacuum to yieldthe product as a colorless crystalline solid (0.795 g, 56.3% yield).Mass calculated m/z 732.5686, observed M⁺¹ (MALDI-TOF ms) m/z 733.8095

Synthesis of propanesulfonate A2-OAc: In a 100 mL round-bottom flaskequipped with a stir bar and reflux condenser, propanesulfonate A2-OH(0.28 g, 0.414 mmol) was dissolved in acetic anhydride (10 mL). Thereaction was heated to 100° C. for 18 h yielding a clear orangesolution, after which time the reaction concentrated in vacuo, andpurified over silica gel (gradient 10% MeOH in DCM to 10% MeOH, 1% sat.NH₄OH in DCM). The product was isolated as an orange sticky solid (211.1mg, 67.0% yield) Mass calculated m/z 760.5999, observed M⁺¹ (MALDI-TOFms) m/z 762.62

Synthesis of propanesulfonate A3-OAc: To a 20 mL vial equipped with astir bar, propanesulfonate A3-OH (0.752 g, 0.673 mmol) and aceticanhydride (10 mL) were added. The vial was sealed and the reactionmixture stirred at 100° C. for 23 h. The reaction mixture was acetoneand concentrated under reduced pressure to yield the crude product as anorange oil (0.91 g, quantitative yield). The crude product was usedwithout further purification. Mass calculated m/z 1283.0516, observedM⁺¹ (MALDI-TOF ms) m/z 1284.74

Synthesis of propanesulfonate A1-OPiv: In a dry 50-mL round bottom flaskequipped with a stir bar and reflux condenser under nitrogen atmophere,was dissolved A1-OH propanesulfonate (0.89 g, 1.37 mmol) indimethylformamide (5 mL), followed by the addition triethylamine (0.96mL, 6.86 mmol, 5 equiv), 4-dimethylamino pyridine (1.7 mg, 0.014 mmol,0.1 equiv) and pivalic anhydride (1.67 mL, 8.23 mmol, 6 equiv). After 14h reaction at 90° C. with stirring, and additional 6 equiv. of pivalicanhydride, 5 equiv. triethylamine, and 0.1 equiv of DMAP was added andthe reaction continued for an additional 26 h, at which point thestarting A1-OH propanesulfonate had disappeared by TLC. The reactionmixture was diluted in dichloromethane, concentrated under reducedpressure, and purified by silica gel chromatography (gravity column)with 10% methanol in dichloromethane to yield the product A1-OPivpropanesulfonate as a sticky brown solid (0.585 g, 52.2%). Masscalculated m/z 817.6625, observed M⁺¹ (MALDI-TOF ms) m/z 817.4124.

Example 3 Activity of the Compositions

The CSALs (FIG. 3) and ZALs (FIG. 16) were formulated into nanoparticlesin the presence of one or more helper lipids such as cholesterol. Asshown in FIG. 17, cholesterol is an important component to allowing thenanoparticles to bind siRNA. In compositions without cholesterol, theamount of siRNA bound was significantly reduced. CSALs nanoparticlesshow an average size of about 100 nm (FIG. 4A). Additionally, thecompositions with increased head group length then the siRNA bindingdecreased (FIG. 4B). Higher mole ratio of CSALs resulted in an increasedcharge (FIG. 4C) and decreased solution pH resulted in higher surfaceparticularly at pH 3 (FIG. 4D). As shown in FIGS. 5 & 6, the CSALsshowed activity in delivering siRNA and thus reducing luciferaseactivity. Additionally, the CSAL containing nanoparticles were testedfor cell viability as well (FIG. 5). Similarly, the ZAL containingnanoparticles were also tested for luciferase activity. (See FIGS.18-20B). Cellular imaging of CSALs and ZALs was carried out to determineif the compositions localized to cells. As shown in FIGS. 7, 21, and 24,the compositions localized to different cells. tRNA delivery was shownin FIG. 22 with a variety of different compositions as delivery of amodified tRNA resulted in restoration of p53 production from a genomewhich contained a nonsense mutation (FIG. 22). Finally, the distributionof the CSALs and ZALs containing nanoparticles in vivo was determined(FIGS. 8 & 23).

The compositions were tested for activity in delivering mRNA and thenucleic acids associated with the CRISPR process such as sgRNA. Thecomposition of the nanoparticles used in these studies is shown below inTables 1A & 1B.

TABLE 1A Zwitterionic Amino Lipid Nanoparticle Molar Compositions ZALMolar ratios in lipid mix ZAL:nucleic Formulation in Mol. PEG acid wtcode formulation Wt. ZAL Cholesterol DSPC DOPE Lipid ratio Z100 ZA3-Ep101191.92 50 38.5 0 0 0 20.00 Z101 ZA3-Ep10 1191.92 50 38.5 0 0 2 20.00Z103 ZA3-Ep10 1191.92 50 38.5 0 0 0.5 20.00 Z102 ZA3-Ep10 1191.92 5038.5 0 0 1 20.00 Z103 ZA3-Ep10 1191.92 50 38.5 0 0 0.5 20.00 Z104ZA3-Ep10 1191.92 40 38.5 10 0 2 15.60 Z105 ZA3-Ep10 1191.92 40 38.5 0 102 15.69 Z106 ZA3-Ep10 1191.92 30 38.5 20 0 2 12.35 Z107 ZA3-Ep10 1191.9230 38.5 0 20 2 12.50 Z108 ZA3-Ep10 1191.92 50 38.5 10 0 2 20.00 Z109ZA3-Ep10 1191.92 50 38.5 0 10 2 20.00 Z110 ZA3-Ep10 1191.92 50 38.5 0 05 20.00 Z111 ZA3-Ep10 1191.92 50 38.5 0 0 10 20.00 Z112 ZA3-Ep10 1191.9250 38.5 0 0 2 10.00 Z113 ZA3-Ep10 1191.92 50 77 0 4 2 10.00 Z114ZA3-Ep10 1191.92 50 38.5 0 0 2 7.50 Z115 ZA3-Ep10 1191.92 50 38.5 0 0 25.00 Z116 ZA3-Ep10 1191.92 50 38.5 0 0 1 10.00 Z117 ZA3-Ep10 1191.92 5038.5 0 0 0.5 10.00 Z118 ZA3-Ep10 1191.92 50 38.5 0 0 1 7.50 Z119ZA3-Ep10 1191.92 50 38.5 0 0 1 5.00 Z120 ZA3-Ep10 1191.92 50 38.5 0 00.5 7.50 Z121 ZA3-Ep10 1191.92 50 38.5 0 0 0.5 5.00 Z122 ZA3-Ep101191.92 50 38.5 0 0 5 10.00 Z123 ZA3-Ep10 1191.92 50 38.5 0 0 5 7.50Z124 ZA3-Ep10 1191.92 50 38.5 0 0 5 5.00 Z125 ZA3-Ep10 1191.92 50 38.5 00 0 10.00 Z202 ZA6-Ep10 933.48 50 38.5 0 0 1 20.00 Z302 ZA3-Ep8 1051.6850 38.5 0 0 1 20.00 Z402 ZA3-Ep12 1332.18 50 38.5 0 0 1 20.00 Z502ZA3-Ep14 1472.48 50 38.5 0 0 1 20.00 Z602 ZA3-Ep16 1612.73 50 38.5 0 0 120.00 Z702 ZA3-Ep18 1753.03 50 38.5 0 0 1 20.00 Z802 ZA1-Ep10 765.2 5038.5 0 0 1 20.00 Z902 ZA4-Ep10 850.35 50 38.5 0 0 1 20.00 Z1002 ZA6-Ac101101.66 50 38.5 0 0 1 20.00 Z1102 ZA6-Ac12 1185.84 50 38.5 0 0 1 20.00

TABLE 1B Cationic Sulfonamide Amino Lipid Nanoparticle MolarCompositions Molar ratios in lipid mix CSAL:nucleic Formulation CSAL inMol. PEG acid wt code formulation Wt. ZAL Cholesterol DSPC DOPE Lipidratio CS100 A3OAcC2Me 1356.19 50 38.5 10 0 1.5 32.1 CS101 A3OAcC2Me1356.19 50 38.5 10 0 1.5 14.3 CS102 A3OAcC2Me 1356.19 50 38.5 10 0 1.521.4 CS103 A3OAcC2M 1356.19 50 38.5 0 0 1.5 20.0 CS104 A3OAcC2M 1356.1950 38.5 0 0 1.5 15.0 CS111 A3OAcC2M 1356.19 50 38.5 0 0 10 15.0 CS200A1OAcC2Me 804.29 50 38.5 10 0 1.5 17.9

Using the ZA1Ep10 ZAL, various concentrations of PEG lipid were testedfor their ability to delivery luciferase mRNA delivery to IGROV1 cells.Monitoring the amount of luminescence produced for each population, theluminescence was measured at 18 hours, 26 hours, and 45 hours posttransfection. The effect of the addition of both the mRNA and the sgRNAin the same particle or sequential administration of these compoundswere tested (FIG. 25). Compositions were the mRNA and the sgRNAgenerally showed reduced amounts of untreated cells relative to the useof different particles. Similar tests were carried out with A549-Luccells (FIG. 26). The effect of the nanoparticle composition with asingle ZAL was tested with HeLa-Luc cells against luciferase and isshown in FIG. 27. As shown, the ZAL result in a dose dependent reductionof luciferase activity (FIG. 28).

Ex vivo imaging of the distribution of the Z120 nanoparticles withdifferent amounts of PEG lipid was analyzed when administered byintravenously and intraperitoneally. BALB-c-Nu mice were injected bytail vein injection with 1 mg/kg Luc mRNA. The mice were then imaged 24hours post injection. These images are shown in FIG. 29. Similar to thedelivery of luciferase, in vivo delivery of Factor VII was analyzedusing Z112 with 3 mg/kg of Factor VII.

Using the CSALs, a dose dependent activity was observed at two differentweight ratio and with two different CSALs (FIG. 30). Using fluorescencelabeled nucleic acids, the internalization of the CSAL nanoparticles wasobserved in A549-luc cells after 24 hour incubation time with 34 nMsiRNA. These images are shown in FIG. 31. Similar imaging was carriedout in BALB-c nude mice showing the internalization of the nanoparticleswithin the body and localization of the nanoparticles into specificorgans as shown in FIG. 32. Binding of suppressor tRNA within the CSALcompositions described herein is shown in FIG. 33 along with particlesize. The gel electrophoresis shows that both CSALs and ZALs when loadedwith suppressor tRNA polymers can restore p53 expression (FIG. 34). Avariety of different ZALs have been shown to be taken up by Calu6 cells(FIG. 35) when loaded with one or more suppressor tRNA molecules.

Example 4 Delivery of CRISPR Nucleotides Using ZALs and CSALs

A. Methods and Materials

i. Chemicals and Reagents for Synthesis.

All chemicals were purchased from Sigma-Aldrich unless otherwiseindicated. 1,2-epoxydecane was purchased from TCI America.1,2-epoxyoctadecane was purchased from Alfa Aesar. Hydrophobic acrylatesoctyl acrylate (Ac8), decyl acrylate (Ac10), tetradecyl acrylate (Ac14),and hexadecyl acrylate (Ac16) were synthesized as described below.Organic solvents were purchased from Fisher Scientific and purified witha solvent purification system (Innovative Technology). Lipid PEG2000 waschemically synthesized, as previously described (Zhou et al., 2016)CDCl₃, methanol-d4, and DMSO-d6 were purchased from Cambridge IsotopeLaboratories.

ii. Nucleic Acids and Other Reagents for Biological Assays.

All siRNAs were purchased from Sigma-Aldrich. DNA oligonucleotides werepurchased from Integrated DNA Technologies. Luciferase, mCherry, andCas9 messenger RNA (mRNA) were purchased from Tri-Link Biotechnologies.Lipofectamine 3000 and OptiMEM were purchased from Invitrogen. Singleguide RNA was prepared by in vitro transcription (IVT) using theMEGAshortscript T7 transcription kit (Life Technologies) followed bypurification using the MEGAclear Transcription Clean-Up Kit (LifeTechnologies) according to the manufacturer's protocols. The Ribogreenreagent was purchased from Life Technologies. ONE-Glo+Tox and Cell TiterGlow were purchased from Promega. RIPA buffer and TRIzol reagent werepurchased from Thermo Scientific. QuickExtract DNA Extraction Solutionwas purchased from Epicentre. Real-time qPCR was performed using iTaqUniversal SYBR Green 2× Supermix (Bio-Rad). All antibodies werepurchased from Cell Signaling.

iii. Cell Culture.

Dulbecco's Modified Eagle Medium (DMEM) was purchased from Hyclonecontaining high glucose, L-glutamine, and without pyruvate or phenolred. RPMI-1640 was purchased from Sigma Aldrich. Dulbecco's modifiedphosphate buffered saline (PBS), Trypsin-EDTA (0.25%) and fetal bovineserum (FBS) were purchased from Sigma-Aldrich. HeLa-Luc and A549-Luccells were cultured in DMEM supplemented with 5% FBS. IGROV1 cells werecultured in RPMI-1640 supplemented with 5% FBS.

iv. Animal Studies.

All experiments were approved by the Institutional Animal Care & UseCommittee (IACUC) of The University of Texas Southwestern Medical Centerand were consistent with local, state and federal regulations asapplicable. C57BL/6 and athymic nude Foxn1^(nu) mice were purchased fromEnvigo. NOD scid gamma (NSG) mice were purchased from the UTSouthwestern animal breeding core. Rosa-CAG-LSL-tdTomato mice werepurchased from The Jackson Laboratory (Stock number: 007909).

v. Methods

¹H and ¹³C NMR were performed on a Varian 400 MHz spectrometer or aVarian 500 MHz spectrometer. MS was performed on a Voyager DE-ProMALDI-TOF. LCMS was performed on an Agilent LCMS system equipped withUV-vis and evaporative light scattering detectors (ELSD). Flashchromatography was performed on a Teledyne Isco CombiFlash Rf-200ichromatography system equipped with UV-vis and evaporative lightscattering detectors (ELSD). Particle sizes and zeta potentials weremeasured by Dynamic Light Scattering (DLS) using a Malvern ZetasizerNano ZS (He—Ne laser, λ=632 nm). RT qPCR was run on a Bio-Rad C1000Touch Thermal Cycler (CFX384 Real-time System). Each reaction was madewith iTaq Universal SYBR Green 2× Supermix (Bio-Rad). Tissue sectionswere imaged using confocal laser scanning microscopy with a ZeissLSM-700 and images were processed using ImageJ (NIH). Flow cytometry wasperformed with BI Fusion machine (BD Biosciences).

vi. Nanoparticle Formulation for in Vivo Studies.

Zwitterionic amino lipid (ZAL) nanoparticles (ZNPs) for in vivo studieswere prepared using a two-channel microfluidic mixer with herringbonerapid mixing features (Precision Nanosystems NanoAssemblr). Ethanolsolutions of lipid mixes (ZALs, cholesterol, and PEG-lipid) were rapidlycombined with acidic aqueous solutions of nucleic acid at an aqueous:EtOH volumetric ratio of 3:1 and a flow rate of 12 mL/minute.

vii. Nucleic Acid Sequences

a. Small Interfering RNAs (siRNAs)

dT are DNA bases. All others are RNA bases.

siLuc (siRNA Against Luciferase).

(SEQ ID NO: 1) sense: 5′-GAUUAUGUCCGGUUAUGUA[dT][dT]-3′ (SEQ ID NO: 2)antisense: 5′-UACAUAACCGGACAUAAUC[dT][dT]-3′

siCtrl (Non-Targeting siRNA)

(SEQ ID NO: 3) sense: 5′-GCGCGAUAGCGCGAAUAUA[dT][dT]-3′ (SEQ ID NO: 4)antisense: 5′-UAUAUUCGCGCUAUCGCGC[dT][dT]-3′

Single guide RNAs (sgRNAs). Guide RNAs were designed using theCRISPR.mit.edu platform and cloned into pSpCas9(BB)-2A-GFP (PX458) aspreviously reported (Ran et al., 2013).

TABLE 2 sgRNA sequences Guide Guide sequence name Target (5′ to 3′) PAMStrand sgLuc1 Luciferase CTTCGAAATGTCCGTTCGGT TGG Positive(SEQ ID NO: 5) sgLuc2 Luciferase CCCGGCGCCATTCTATCCGC TGG Positive(SEQ ID NO: 6) sgLuc3 Luciferase TCCAGCGGATAGAATGGCGC CGG Negative(SEQ ID NO: 7) sgLuc4 Luciferase GGATTCTAAAACGGATTACC AGG Positive(SEQ ID NO: 8) sgLuc5 Luciferase ATAAATAACGCGCCCAACAC CGG Negative(SEQ ID NO: 9) sgLoxP LoxP CGTATAGCATACATTATACG AAG Negative(SEQ ID NO: 10) sgCtrl Mouse F7 GCTTCGATAATATCCGCTAC TGG Positive(SEQ ID NO: 11)

TABLE 3 BbsI sgRNA cloning oligos SEQ ID Probe Sequence (5′ to 3′)* NO:sgLuc1_Top CACCGCTTCGAAATGTCCGTTCGGT 12 sgLuc1_BottomAAACACCGAACGGACATTTCGAAGC 13 sgLuc2_Top CACCGCCCGGCGCCATTCTATCCGC 14sgLuc2_Bottom AAACGCGGATAGAATGGCGCCGGGC 15 sgLuc3_TopCACCGTCCAGCGGATAGAATGGCGC 16 sgLuc3_Bottom AAACGCGCCATTCTATCCGCTGGAC 17sgLuc4_Top CACCGGGATTCTAAAACGGATTACC 18 sgLuc4_BottomAAACGGTAATCCGTTTTAGAATCCC 19 sgLuc5_Top CACCGATAAATAACGCGCCCAACAC 20sgLuc5_Bottom AAACGTGTTGGGCGCGTTATTTATC 21 sgLoxP_TopCACCGCGTATAGCATACATTATACG 22 sgLoxP_Bottom AAACCGTATAATGTATGCTATACGC 23sgCtrl_Top CACCGGCTTCGATAATATCCGCTAC 24 sgCtrl_BottomAAACGTAGCGGATATTATCGAAGCC 25 *Guide sequence shown in bold.

TABLE 4 T7 template PCR primers SEQ ID Primer Sequence (5′ to 3′) NO:IVT sgLuc- TAATACGACTCACTATAGGGATAAATAACGCG 26 fwd CCCAACAC IVT sgLoxP-TAATACGACTCACTATAGGGCGTATAGCATAC 27 fwd ATTATACG IVT sgCtrl-TAATACGACTCACTATAGGGGCTTCGATAATA 28 fwd TCCGCTAC IVT-rev AAAAGCACCGACTCGGTGCC 29 (common)

TABLE 5 Surveyor assay PCR primers Expected Ampli- cut PrimerSequence (5′ to 3′) con bands Luc 1_ GGAACCGCTGGAGAGCAACT 510 bp233 bp,  Forward (SEQ ID NO: 30) 277 bp Luc 1_ GTCCCTATCGAAGGACTCTGGCAReverse (SEQ ID NO: 31) Luc 2_ GCTGGAGAGCAACTGCATAA 429 bp 202 bp, Forward (SEQ ID NO: 32) 227 bp Luc 2_ CATCGACTGAAATCCCTGGTAATC Reverse(SEQ ID NO: 33)

TABLE 6 Real time qPCR primers SEQ ID Primer Sequence (5′ to 3′) NO:Cas9 forward GGAACCGCTGGAGAGCAACT 34 Cas9 reverseGTCCCTATCGAAGGACTCTGGCA 35 hActinB forward AGAAGGATTCCTATGTGGGCG 36hActinB reverse CATGTCGTCCCAGTTGGTGAC 37

viii. sgRNA Preparation.

Single guide RNAs were designed using the CRISPR.mit.edu platform andcloned into PX458 plasmid with standard Bbsl cloning. T7 transcriptiontemplates were amplified by PCR and gel purified. sgRNAs weresynthesized by in vitro transcription using the MEGAshortscript T7transcription kit (Life Technologies) followed by purification using theMEGAclear Transcription Clean-Up Kit (Life Technologies) according tothe manufacturer's protocols.

ix. Screening of sgRNA Using pDNA.

sgRNA-cloned PX458 plasmids were used to evaluated efficacy of thesgRNAs against luciferase by transfection of the plasmid encoding bothsgRNA and Cas9. Lipofectamine 3000 (LF3000, Invitrogen) was used totransfect the sgRNA-Cas9 plasmids according to manufacturer's protocols.HeLa-Luc cells were seed in a 96-well white-opaque tissue culture plateat a density of 10,000 cells per well. LF3000 pDNA particles were addedto the cells at a dose of 100 ng pDNA per well. After 6 hours, themedium was removed and exchanged for 200 μL fresh growth medium. After24, 48 and 72 h, the relative expression of luciferase was determinedusing the One-Glo+Tox assay (Promega) and normalized to control.Non-targeting sgRNA (sgScr) and unguided Cas9 plasmids were used as acontrol. (N=4+/− standard deviation).

x. HeLa-Luc-Cas9 Cell Line Preparation.

HeLa-Luc-Cas9 stable cells were prepared by lentiviral transduction.Parental HeLa-Luc cells (Zhou et al., 2016; Hao et al., 2015) wereseeded at a density of 70,000 cells per well in a 24-well plate incomplete growth medium and allowed to attach in the incubator overnight.The medium was replaced with 1 mL pre-warmed pseudoparticle medium(DMEM, 3% FBS, 20 mM HEPES, 4 μg/mL polybrene). Cas9-Blast lentivirussupernatant was thawed on ice and 50-100 μL was added to the desiredwell. The cells were spinoculated at room temperature for 1 hour at1,000×g, and returned to the incubator overnight, after which thepseudoparticle medium was exchanged for complete growth medium. After 48h total time post spinoculation, selective pressure was applied (5 and10 μg/mL Blasticidin S) and cells were maintained and expanded. Singlecell clones were isolated by single cell sorting by flow cytometry. Cas9protein expression was confirmed by western blot compared to parentalHeLa-Luc cells by blotting for FLAG tag before single cell sorting andfor Cas9 after single cell sorting.

xi. In Vitro ZAL Nanoparticle (ZNP) Formulations.

ZNPs were prepared by the ethanol dilution method. The RNA (whether ansiRNA, sgRNA, or mRNA) was diluted in acidic aqueous buffer (unlessotherwise indicated, 10 mM citric acid/sodium citrate buffer pH 3). Thelipid mix was prepared in ethanol, with the appropriate molar ratios ofZAL, cholesterol and PEG-lipid from ethanol stock solutions of eachcomponent. Via pipette, the lipid dilution was added to the RNA dilutionat a final volumetric ratio of 1:3, rapidly mixed by pipette, andincubated for 15-20 minutes. After this incubation period, the particleswere either diluted 3-fold in, or dialyzed against 1× Dulbecco'sModified PBS without calcium and magnesium (Sigma-Aldrich). Dialyseswere performed in Pur-A-Lyzer Midi dialysis chambers (Sigma-Aldrich) for1 hour per 200 μL sample per chamber.

xii. ZAL siRNA Delivery Library Screen.

The library of ZALs functionalized with epoxide and acrylate hydrophobictails was screened for siRNA delivery efficacy in HeLa-Luc cells. In awhite opaque 96-well plate tissue culture plate, HeLa cells were seededat a density of 10×10³ cells per well in 100 μL growth medium (DMEMwithout phenol red, 5% FBS), and allowed to attach overnight. The mediumwas exchanged for 200 μL fresh growth medium the day of the assay. CrudeZALs products were prepared using a formulation lipid mixture of 50:38.5(ZAL: cholesterol), and a ZAL:siRNA ratio such that the number ofhydrophobic tails in the ZAL times the ZAL:siRNA mole ratio in theformulation was ˜1000, which resulted in a weight ratio range across thelibrary of 16:1 to 45:1 ZAL:siRNA, with an average of 29.5+/−6.3 weightratio across the library. ZAL NP formulations were performed in a96-well plate by rapid mixing of ZAL lipid mix (20 μL) and siLucdilution (60 μL, 13.33 ng/μL in 10 mM citric acid-sodium citrate buffer,pH 5) at 3:1 aqueous:EtOH v:v ratio with a multichannel pipette. After a15-20 minute incubation period, the formulations were diluted in 12volumes (240 μL) PBS. The nanoparticles (40 μL) were added to theHeLa-Luc cells at a dose of 100 ng siRNA per well. The nanoparticleswere incubated with the cells for 24 h after which time the cellviability and luciferase expression were evaluated with the ONE-Glo+ToxAssay cell viability and luciferase assay (Promega).

xiii. sgRNA Delivery to HeLa-Luc-Cas9 Cells.

Select ZALs were evaluated in the delivery of single guide RNA (sgRNA)to HeLa-Luc-Cas9 cells. In a white opaque 96-well plate tissue cultureplate, HeLa-Luc-Cas9 cells were seeded at a density of 5×10³ cells perwell in 100 μL growth medium (DMEM without phenol red, 5% FBS), andallowed to attach overnight and then supplemented with an additional 100μL DMEM. ZNPs encapsulating sgRNA were formulated using the in vitronanoparticle formulation protocol at the indicated lipid composition andweight ratio (maintaining 50:38.5 (ZAL:cholesterol mole ratio), tuningPEG-lipid additive from 5% to 0.5%, and tuning weight ratio from 20:1ZAL:sgRNA to 5:1 ZAL:sgRNA). Non-targeting control sgRNA (sgCtrl) wasused as a negative control. The nanoparticles were added to the cells atthe appropriate dose of sgRNA and incubated with the cells for 48 h. Thecell viability and luciferase expression were evaluated with theONE-Glo+Tox Assay (Promega), normalized to untreated cells (N=4+/−standard deviation).

xiv. Kinetic Assay of sgRNA and siRNA Delivery.

The kinetics of luciferase expression after silencing/editing by siRNAand sgRNA were determined in HeLa-Luc-Cas9 cells. For time points <48 h,ZNPs encapsulating sgRNA or siRNA were delivered to HeLa-Luc-Cas9 cellsin 96-well plates at a density of 5K cells per well. After 0.5, 1, 2, 4,11, 20, 30 and 44 h time point, the cell viability and luciferaseexpression were determined by the One-Glow+Tox assay. For longer timepoints, cells were treated in 6-well plates. Beginning at the 2 day timepoint, cells were aspirated, washed with 1× PBS, trypsinized in 200 μLtrypsin and re-suspended in 1800 μL medium. 1 mL of each cell suspensionwas added to a fresh 6-well plate containing 1 mL DMEM (2 mL total) andreturned to the incubator. Of the remaining cell suspension, 50 μL wastransferred to a 96-well white-opaque plate (10 wells per sample). Cellviability was determined using the Cell-Titer Glo assay normalized tountreated cells, while relative luciferase expression was determinedusing the One-Glo assay and normalized against control (siCtrl orsgCtrl). Data was plotted as an average of 5 measurements+/− standarddeviation.

xv. Luciferase mRNA Delivery in Vitro Assay.

ZNPs with mRNA (Tri-Link Biotechnologies) were prepared using the invitro nanoparticle formulation method outlined above. IGROV1 cells wereseeded in white opaque 96-well tissue culture plates at a seedingdensity of 5×10³ cells per well in 100 μL RPMI 1640 medium supplementedwith 5% FBS, and allowed to attach overnight. After overnightincubation, an additional 100 μL medium was added to the wells. TheZAL:mRNA nanoparticles were prepared at ZAL:mRNA weight ratios of 20:1,10:1, 7.5:1 and 5:1, and lipid mixture molar compositions of 50:38.5:nZAL:cholesterol:PEG-lipid, where n=5, 2, 1, and 0.5 at each weightratio. The ZAL-mRNA nanoparticles were added to the cells at theappropriate mRNA dose and incubated for the indicated time (ranging from6 h to 48 h), after which time cell viability and luciferase expressionwere evaluated with the ONE-Glo+Tox Assay (Promega) and normalized tountreated cells (N=4+/− standard deviation).

xvi. In Vitro Co-Delivery of Cas9 mRNA and sgRNA.

ZNPs were evaluated in the co-delivery of Cas9 mRNA (Tri-Linkbiotechnologies) and single guide RNA (sgRNA) to luciferase expressingcancer cells. Cells were seeded at a density of 250,000 per well in6-well plates and 2-mL DMEM. ZNPs were formulated using the in vitroformulation protocol. For co-delivery in a single particle, Cas9 mRNAand sgRNA were combined in acidic buffer together at pH 3 prior to theaddition of ZAL lipid mix at the appropriate ZAL:total RNA weight ratio.Cells were incubated with ZNPs for 72 h prior to evaluation of editingby the surveyor assay. As a negative control, ZNPs with Cas9 only(unguided Cas9), sgLuc only, and Cas9 plus sgCtrl were added. sgRNA dosewas fixed at 0.5 μg per well, while Cas9 mRNA dose was tuned from 0.5 μg(1:1) to 3 μg (6:1) per well. ZAL:total RNA ratio was fixed at 7.5:1.Staged co-delivery was carried out by the addition of Cas9 mRNA ZNPsfollowed by the addition of sgRNA ZNPs 24 h later at a total ratio of2:1 Cas9 mRNA to sgRNA. Following an additional 48 h incubation time,cells were evaluated by gene editing by the surveyor assay.

xvii. Nucleic Acid Binding Experiments.

Nucleic acid binding was evaluated using the Ribogreen assay (MolecularProbes). In short, nanoparticles were prepared using the in vitro or invivo formulation protocols. The nanoparticle formulations (5 μL) wereadded to a black 96-well opaque microplate (Corning). A standard curveof the appropriate nucleic acid was prepared in the same medium as thenanoparticles. Ribogreen reagent was diluted 1:1000 in 1× PBS and 50 μLwas added to each well via multichannel pipette. The mixture was stirredon an orbital mixer for 5 minutes, and the fluorescence of each well wasread using a plate reader (λ_(Ex) 485 nm, λ_(Em) 535 nm). The amount offree nucleic acid was determined by fitting the signal from eachnanoparticle sample to the nucleic acid standard curve, and the fractionbound determined by the following formula: Fraction nucleic acidbound=(total nucleic acid input-free nucleic acid)/total nucleic acidinput) (N=3 or 4+/− standard deviation).

xviii. In Vivo Nanoparticle Formulations:

In vivo nanoparticle formulations were performed using the NanoAssemblrmicrofluidic mixing system (Precision Nanosystems). Lipids weredissolved in ethanol and nucleic acids were diluted in 10 mM citricacid-sodium citrate buffer pH 3. The lipid mixture and nucleic aciddilution were combined at a volumetric ratio of 3:1 nucleic acid:lipidmix at a total flow rate of 12 mL per minute, and a waste collection of0.1 mL at the start and end of each formulation. The nanoparticles weredialyzed against 1× PBS in Pur-A-Lyzer midi dialysis chambers(Sigma-Aldrich) for 1 hour per 200 μL volume in each chamber, anddiluted in 1× PBS to the appropriate nucleic acid concentration.

xix. In Vivo Luciferase mRNA Delivery:

All experiments were approved by the Institutional Animal Care & UseCommittee (IACUC) of The University of Texas Southwestern Medical Centerand were consistent with local, state and federal regulations asapplicable. ZA3-Ep10 was formulated with in vivo formulation at 50ZAL:38.5 cholesterol: 0.5, 1, or 2 PEG-lipid mole ratio in the lipidmix, and 7.5:1 ZAL:mRNA weight ratio. Mice were injected with ZAL-mRNANPs at a dose of 1 mg/kg via tail vein injection or intraperitonealinjection. After 24 h and 48 h the luciferase expression was evaluatedby live animal bioluminescence imaging Animals were anesthetized underisofluorane, and D-luciferin monosodium hydrate (GoldBio) substrate wasinjected subcutaneously in the neck scruff. After 10-12 minuteincubation under anesthesia, the luciferase activity was imaged on anIVIS Lumina system (Perkin Elmer), and the images processed using LivingImage analysis software (Perkin Elmer). Ex vivo imaging was performed onsystemic organs after resection, and the tissue frozen on dry ice for exvivo luciferase expression analysis.

xx. Nanoparticle Property Characterization

Physical properties were measured using a Zetasizer Nano ZS (Malvern)with an He—Ne laser (λ=632 nm). Particle sizes were measured by dynamiclight scattering (DLS) (5 measurements, 3 runs×10 seconds, automaticattenuator setting) by 173° back scattering. Zeta potential was measuredin a folded capillary cell (Malvern) with samples diluted in PBS for ZALNPs or citrate phosphate buffer pH 7.4 for CSAL NPs.

xxi. Surveyor Assay

Genomic DNA from transfected cells was isolated using QuickEx DNAExtraction Solution (Thermo Fisher Scientific) according to themanufacturer's protocol. Then the target region was amplified by PCR,and the PCR products were gel purified on an agarose gel (QIAquick GelExtraction Kit, QIAgen). Surveyor assay was perforated using SurveyorMutation Detection Kit (IDT): the PCR products were first hybridized,then half of the products were cut with Nuclease S; both the uncut andcut DNA were then run on the 4-20% polyacrylamide gel (Biorad). The gelswere stained with SYBR Gold Nucleic Acid Gel Stain buffer (diluted1:10000 in TBE buffer, Thermo Fisher Scientific) and imaged by UV light.

xxii. Western Blot

The cells were lysed in cold RIPA buffer (Thermo Scientific), the lysatecleared by centrifugation and total protein in the supernatantquantified by the BCA assay (Pierce). 50 μg total protein was loaded on4-20% precast polyacrylamide gel and transferred to a nitrocellulosemembrane (BioRad). The membrane was blocked in 5% nonfat milk for 1 hourat RT, and then incubated with primary antibody at 4° C. overnight (Cas9antibody, 1:1000, Cell Signaling, 146975; beta-actin antibody, 1:2000,Cell Signaling, 4970). Secondary antibodies were applied at RT for 1hour (anti-rabbit IgG, HRP-linked antibody, Cell Signaling, 7074,anti-mouse IgG, HRP-linked antibody, Cell Signaling, 7076), and then themembrane was developed and detected on X-ray film.

xxiii. Real-Time RT-qPCR.

Cells were transfected with Cas9 mRNA for the indicated time point in a6-well plate and 0.5 μg/mL mRNA for the indicated time point. Total RNAwas extracted using the TRIzol reagent according to the manufacturer'sprotocol. The RNA was reverse transcribed using the iScript ReverseTranscription kit (BioRad) and the real-time qPCR was run on a Bio-RadC1000 Touch Thermal Cycler (CFX384 Real-time System). Each reaction wasmade with iTaq Universal SYBR Green 2× Supermix (Bio-Rad). The qPCRprogram is as follows:

1) 95° C. for 3 min

2) 95° C. 10 s and 55° C. 30 s for 40 cycles

3) 95° C. 10 s

4) 65° C. 5 s

5) 95° C. 5 s

Human β-actin was used as a control and mRNA levels were normalized tofold actin and plotted as an average of two independent experiments.

xxiv. In Vivo Delivery of Cas9 mRNA and sgLoxP.

ZA3-Ep10 ZNPs encapsulating Cas9 mRNA and sgLoxp were prepared accordingto the in vivo nanoparticle formulation protocol using the Nanoassemblrmicrofluidic mixing device. The lipid mix contained 50 ZA3-Ep10: 38.5cholesterol: 0.5 PEG-lipid molar ratios, and the particles wereformulated at a 7.5:1 ZAL:total RNA weight ratio. The Cas9 mRNA: sgLoxPweight ratio was maintained at 4:1. Rosa 26-LSL-tdTomato mice wereinjected at 5 mg/kg total RNA (4 mg/kg mRNA, 1 mg/kg sgRNA) via tailvein injection and monitored for 1 week. After which they weresacrificed and the major organs imaged using the IVIS Lumina system forfluorescence expression (dsRed filter set) compared to an uninjectedRosa 26-LSL-tdTomato mouse. A liver specific Cre recombinaseadeno-associated virus (Cre-AAV8) injected intravenously via tail veininjection (4 days) was used as a positive control.

xxv. Tissue Sectioning

Tissue were fixed in 4% paraformaldehyde (PFA) at RT for 2 hours, thenchanged in 30% sucrose (in PBS) at 4° C. overnight. Then the tissueswere embedded in Cryo-gel (Leica Biosystems), and frozen in dry ice. Theblocks were sectioned using Cryostat machine (Leica Biosystems) at 8 μmthickness. The sections were air-dried and incubated in 0.25% TritonX-100 (Biorad) 5% FBS in PBS for 1 h at RT, Then the slides were mountedwith DAPI (Vector Laboratories) and covered,

xxvi. Primary Hepatocytes Isolation

Primary hepatocytes were isolated by two-step collagenase perfusion.Liver perfusion medium (Thermo Fisher Scientific, 17701038), liverdigest medium (Thermo Fisher Scientific, 17703034) and Hepatocytes washmedium (Thermo Fisher Scientific, 17704024) were used.

xxvii. Flow Cytometry

For detection of Tomato positive populations, primary hepatocytes(2×1.0⁶/mL) were isolated and stained with DAPI (Roche, 2 μg/mL) fordead cell exclusion. Cells were analyzed with BD FACSAria Fusion machine(BD Biosciences). Tomato positive cells were counted in DAPI negative(live cell) populations.

xxviii. Statistical Analysis

Statistical analysis was performed using a Student's t-test in GraphPadPrism.

B. Delivery of CRISPR Nucleic Acid Sequences

Zwitterionic amino lipids (ZALs) were rationally synthesized to containa zwitterionic sulfobetaine head group, an amine rich linker region, andassorted hydrophobic tails (FIG. 67). A zwitterionic electrophilicprecursor (SBAm) was prepared by the ring-opening reaction of2-(dimethylamino)ethyl acrylamide with 1,3-propanesultone, which waseasily isolated by in situ precipitation in acetone. Conjugate additionof different polyamines to SBAm afforded a series of zwitterionic aminesthat could be reacted with hydrophobic epoxides and acrylates to append6 to 18 carbon alkyl tails and alcohol/ester groups to enhance ZAL-RNAinteractions (See Example 2). To verify that ZNPs could generally bindand deliver RNA, the 72-member library was first screened for siRNAdelivery to HeLa cells that stably expressed firefly luciferase(HeLa-Luc) (FIG. 19). This allowed structural identification of keyamine cores, including ZA1, ZA3, and ZA6. Interestingly, epoxide-basedZALs (ZA_(x)-Ep_(n)), were also generally more active thanacrylate-based ZALs (ZA_(x)-Ac_(n)) (FIG. 18). With lead compounds inhand focus turned to the delivery of sgRNAs and Cas9 mRNA. Bothtemporally staged and simultaneous co-delivery enabled fully exogenousgene editing.

ZALs were evaluated for their ability to deliver CRISPR/Cas9 componentsusing a stable cell line expressing both Cas9 and luciferase(HeLa-Luc-Cas9). A single HeLa-Luc-Cas9 cell clone was isolatedfollowing Cas9 lentiviral transduction of HeLa-Luc cells (FIGS.43A-43C). sgRNAs against luciferase were designed and generatedaccording to previously reported methods targeting the first third ofthe gene (Table 2) (Ran et al., 2013) and evaluated by pDNA transfection(FIG. 44). The most active sgRNA against luciferase (sgLuc5, henceforthsgLuc) as well as control sgRNAs were synthesized by in vitrotranscription. Next, lead ZNPs were loaded with sgLuc and evaluated fordelivery to HeLa-Luc-Cas9 cells. Luciferase and viability (Hao et al.,2015; Zhou et al., 2016; Yan et al., 2016) were measured after 48 hours(h) relative to untreated cells. As anticipated from the chemical designcombining cationic and zwitterionic functionalities, ZNPs do not requireinclusion of helper phospholipids (FIG. 65A).

Among the lead ZALs, ZA3-Ep10 was found to be an efficacious fordelivery of sgLuc (FIG. 45). Editing of luciferase DNA resulted in adose-dependent decrease in luciferase expression (FIG. 65B). CRISPR/Casediting were verified using the Surveyor nuclease assay, (Guschin etal., 2010) which can detect indels (FIG. 63C). Given that sgRNAs requireloading into Cas9 nucleases in cells and trafficking to the nucleus toperform sequence-guided editing, understanding of the kinetics of thisprocess was sought, particularly in comparison to RNAi-mediated genesilencing. siLuc-mediated mRNA degradation is a fast process, whereexpression decreased by 40% within the first 4h. Luciferase was silencedby 92% by 20 h and remained low for about 3 days. Thereafter, theprotein expression steadily increased and reached baseline level 6 daysafter transfection (FIG. 63B and FIG. 46 (early time points)). Incontrast, sgLuc-mediated DNA editing was kinetically slower, possiblydue to the requirements to load into Cas9 and survey the DNA for PAMs.It took 20 h for luciferase expression to decrease by 40%, ultimatelygoing down by 95% after 2 days and remaining there indefinitely. The lowluciferase expression (5%) persisted throughout the duration of theassay (9 days) due the permanent genomic change, even after multiplerounds of cellular division, suggesting that edited cells grew at thesame rate of non-edited cells (FIGS. 63B and 47).

Having demonstrated that ZA3-Ep10 ZNPs could effectively deliver sgRNAs(˜100 nt), their ability to deliver even longer mRNA (1,000 to 4,500 nt)was examined next. mRNA encoding mCherry mRNA (˜1,000 nt) or luciferasemRNA (˜2,000 nt) was delivered to IGROV1 human ovarian cancer cells.Bright mCherry expression was visible (FIG. 63C), and luciferaseexpression was observed to be dose-dependent (FIG. 63D). Notably, highexpression required low mRNA doses (<600 pM). In contrast to sgRNA,which did not show a dependence on PEG lipid mole ratio in theformulation (FIGS. 28 & 48), delivery efficacy of mRNA decreased withhigher PEG lipid ratios (FIG. 49), while there was only a modest changein ZNP size (FIG. 50). Optimization of PEGylation, particularly in viewof in vitro to in vivo translation, is an ongoing challenge to beexplored for each target disease, organ, and cell type (Whitehead etal., 2012). This report attempts to alleviate some of those concerns byexamining different formulations in multiple cell types and mousestrains. Further supporting the design hypothesis, titration of astructurally analogous cationic lipid with increasing molar proportionsof DOPE into the formulations showed an improvement in delivery of sgRNAand mRNA, while siRNA did not require additional zwitterionic content(FIG. 51). Moreover, efficacy of ZA3-Ep10 ZNPs was consistent across allRNA cargos, and outperformed the cationic analogue supplemented withphospholipid.

The optimal formulation was next evaluated in vivo through intravenous(i.v.) administration of ZA3-Ep10 mRNA ZNPs to multiple strains of mice.Bioluminescence imaging following Luc mRNA delivery in athymic nude mice(FIG. 65E, 1 mg/kg), C57BL/6 mice (FIG. 65F, 4 mg/kg), and NOD scidgamma (NSG) mice (FIG. 52, 1 mg/kg) resulted in expression of luciferasein liver, lung and spleen tissue 24h after injection which wasquantified by ROI analysis (FIGS. 53A & 53B). Based on the high lungsignal, co-delivery (one pot) CRISPR/Cas editing in lung cells wasexplored.

Due the very long length of Cas9 mRNA (4,500 nt), delivery usingsynthetic carriers is particularly challenging. Remarkably, the level ofCas9 mRNA in A549 lung cancer cells was found to be very high after only4 h incubation with ZA3-Ep10 Cas9 mRNA ZNPs (FIG. 66A). Syntheticallyintroduced mRNA decreased from >4 fold actin to 0.7 fold actin over thenext 45 h. Because translation of mRNA takes time, protein expressionwas low at 4 h, increased considerably by 12 h, and was the highest by36 h (FIGS. 66A & 66B). It was also dose dependent (FIG. 66C). For invivo utility, the use of synthetic NP carriers alleviates concerns ofviral delivery. Moreover, delivery of Cas9 mRNA allows for transientexpression of Cas9, minimizing persistence that can lead to off-targetgenomic alteration. This can reduce the significant therapeutic dangerof incorporating an exogenous nuclease into the genome.

As illustrated above, delivery of mRNA and sgRNA is kineticallydifferent. Indeed, it was found that staged delivery in separate ZNPswas an effective treatment method. ZNP delivery of mRNA for 24 h, toenable Cas9 protein expression, followed by sgRNA delivery in separateZNPs enabled efficacious in vitro editing in both HeLa-Luc and A549-Luccells (FIGS. 54 & 55). However, when considering in vivo utility, Cas9mRNA and sgRNA must be present in the same cell. It was thereforereasoned that co-delivery of mRNA and sgRNA from a single NP wouldprovide a greater editing efficiency since this method would guaranteedelivery to the same individual cells. A variety of conditions wereexplored and found that effective editing of the target gene by ZNPsencapsulating both Cas9 mRNA and sgRNA required a ratio of mRNA:sgRNAgreater than or equal to 3:1 (wt) as confirmed by the Surveyor assay(FIG. 66D), while control ZNPs did not show any editing (FIG. 55).

To examine co-delivery in vivo, genetically engineered mice containing ahomozygous Rosa26 promoter Lox-Stop-Lox tdTomato (tdTO) cassette presentin all cells were utilized (Tabebordbar et al., 2016). Co-delivery ofCas9-mRNA and sgRNA against LoxP (Li et al., 2015) enabled deletion ofthe Stop cassette and induction of tdTO expression (FIG. 67A, Table 2).This is a challenging model for a synthetic carrier due to the need tomake two cuts on the same allele for the tdTO to be expressed. ZNPsencapsulating Cas9 mRNA and sgLoxp at a 4:1 mRNA:sgRNA weight ratio wereadministered intravenously at a 5 mg/kg RNA dose (FIGS. 56-58). One weekafter administration, fluorescence signal from tdTO was detected in theliver and kidneys upon whole organ ex vivo imaging (FIG. 67B). Detailedexamination of sectioned organs using confocal fluorescence microscopyshowed tdTO-positive cells in liver, lung, and kidney tissues (FIG.67C). Importantly tdTO positive cells were not detected when animalswere treated with sgCtrl ZNPs (FIG. 59) and no significant change inbody weight of treated animals was observed (FIG. 60). Primaryhepatocytes were isolated from perfused livers and tdTO cells werecounted by flow cytometry to quantify editing (FIG. 61). To furtherconfirm editing, tissues were harvested 2 months after ZNP sgLoxPtreatment, which still exhibited strong fluorescent signal in the liverand kidneys (FIG. 62). This proof-of-principle data indicates thatintravenous co-delivery of Cas9 mRNA and targeted sgRNA from a singleZNP can enable CRISPR/Cas editing in vivo.

Example 5 Further Modification of ZALs and CSALs

Given the modular nature of the synthesis of the ZALs and CSALs, changeswithin a single portion of the molecule can be effected withouthampering the ability to obtain a large variety of structural analogs.For example, the central cationic amine of the ZALs and CSALs can bemodified as shown below to obtain different length chains.

Additionally, the anionic headgroup of the ZALs may be replaced with adifferent anionic group or the converted to a cationic head group whenthe central amine has been replaced with an anionic phosphate group asshown below.

Analysis of these groups will modify sterics, zwitterionic identify andspacing of the charges within the molecule. These modified anionic headgroups may be synthesized as described in the Scheme below.

Additionally, the formation of the hydrophobic tails using theepoxidized starting materials results in the presence of a secondaryalcohol site which would be further reacted to generate additionalinteractions with the nucleic acid sequences. Some non-limiting examplesof possible modifications include those shown in the Scheme below. TheNMR of the acetylated ZAL is shown in FIG. 68.

Additionally, the amines can be functionalized with a degradable diestersuch as the one shown below. This diester can be further modified withone or more mercapto alkyl groups to provide the necessarily hydrophobicgroups.

Furthermore, to allow for the introduction of both degradable estergroups as well as a secondary alcohol, glycidic esters were prepared.

Formation of these compounds with different acrylates was carried outand shown via NMR in FIGS. 69A & 69B. These modified structural ZALs andCSALs were tested for their ability to bind RNA as well as the physicalproperties of nanoparticles formed with different RNA molecules. Theseparticles were then tested for their ability to delivery a sgRNA to aHeLa-Luc-Cas9 cell and an mRNA to an IGROV1 cell. The mass spectral andNMR data for additional analogs is shown in FIGS. 70 and 71,respectively.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of certain embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the disclosure as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A compound of the formula:

wherein: X₁ is —S(O)₂O⁻, —OP(O)OR_(e)O⁻, —(CHR_(f))_(z)C(O)O⁻, or—NR_(g)R_(h)R_(i) ⁺, wherein: R_(e), R_(g), R_(h), and R_(i) are eachindependently hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6));R_(f) is hydrogen, amino, hydroxy, or alkyl_((C≦12)), aryl_((C≦12)),aralkyl_((C≦12)), heteroaryl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),acyloxy_((C≦12)), amido_((C≦12)), alkoxy_((C≦12)), alkoxy_((C≦12)), or asubstituted version of any of the last ten groups; and z is 1, 2, 3, or4; Y₁ is alkanediyl_((C≦12)), alkenediyl_((C≦12)), arenediyl_((C≦12)),heteroarenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),-alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12)),-alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),-alkane-diyl_((C≦8))-heteroarenediyl_((C≦12)),-alkanediyl_((C≦8))-heteroarene-diyl_((C≦12))-alkanediyl_((C≦8)), or asubstituted version of any of these groups; Z₁ is —N⁺R₃R₄— or —OP(O)O⁻O—A is —NR_(a)—, —S—, or —O—; wherein: R_(a) is hydrogen, alkyl_((C≦6)),or substituted alkyl_((C≦6)), or R_(a) is taken together with either R₃or R₄ and is alkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),alkylaminodiyl_((C≦8)), or a substituted version of any of these groups;R₁ is a group of the formula:

wherein: R₅, R₆, and R₂ are each independently hydrogen oralkyl_((C≦8)), -alkanediyl_((C≦6))NH₂,-alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), -alkanediyl_((C≦6))-NR′R″, ora substituted version of any of these groups wherein: R′ and R″ are eachindependently hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or—Z₂A′R₇; wherein: Z₂ is alkanediyl_((C≦6)), substitutedalkanediyl_((C≦6)), or a group of the formula:

wherein:  Z₅ and Z₆ are each independently alkanediyl_((C≦6)) orsubstituted alkanediyl_((C≦6));  X₂ and X₃ are each independently —O—,—S—, or —NR_(m)—; wherein:   R_(m) is hydrogen, alkyl_((C≦6)), orsubstituted alkyl_((C≦6)); and  a is 0, 1, 2, 3, 4, 5, or 6; A′ is—CHR_(j)—, —S—, —C(O)O—, or —C(O)NR_(b)—;  R_(b) is hydrogen,alkyl_((C≦6)), or substituted alkyl_((C≦6)); and  R_(j) is hydrogen,halo, hydroxy, acyloxy_((C≦24)), or substituted acyloxy_((C≦24)); R₇ isalkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)); or R₅, R₆, and R₂ are each independently—Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦6)), substitutedalkanediyl_((C≦6)), or a group of the formula:

wherein:  Z₇ and Z₈ are each independently alkanediyl_((C≦6)) orsubstituted alkanediyl_((C≦6));  X₄ and X₅ are each independently —O—,—S—, or —NR_(n)—; wherein:   R_(n) is hydrogen, alkyl_((C≦6)), orsubstituted alkyl_((C≦6)); and  b is 0, 1, 2, 3, 4, 5, or 6; A″ is—CHR_(k)—, —S—, —C(O)O—, or —C(O)NR_(l)—;  R_(l) is hydrogen,alkyl_((C≦6)), or substituted alkyl_((C≦6)); and  R_(k) is hydrogen,halo, hydroxy, acyloxy_((C≦24)), or substituted acyloxy_((C≦24)); and R₈is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)); q is 1, 2, or 3; and r is 1, 2, 3, or 4;R₁ is a group of the formula:

wherein: Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a substituted versionof any of these groups; R₉, R₁₀, and R₁₁ are each independentlyhydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or —Z₄A′″R₁₂;wherein: Z₄ is alkanediyl_((C≦6)), substituted alkanediyl_((C≦6)), or agroup of the formula:

wherein:  Z₉ and Z₁₀ are each independently alkanediyl_((C≦6)) orsubstituted alkanediyl_((C≦6));  X₆ and X₇ are each independently —O—,—S—, or —NR₀—; wherein:   R₀ is hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)); and  c is 0, 1, 2, 3, 4, 5, or 6; A′″ is —CHR_(k)—, —S—,—C(O)O—, or —C(O)NR_(l)—;  R_(l) is hydrogen, alkyl_((C≦6)), orsubstituted alkyl_((C≦6)); and  R_(k) is hydrogen, halo, hydroxy,acyloxy_((C≦24)), or substituted acyloxy_((C≦24)); and R₁₂ isalkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)); and x and y are 0, 1, 2, 3, or 4; R₃ andR₄ are each independently hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)), or R₃ or R₄ are taken together with R_(a) and isalkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),alkylaminodiyl_((C≦8)), or a substituted version of any of these groups;and m, n, and p are each independently an integer selected from 0, 1, 2,3, 4, 5, or 6; provided that if X₁ is positively charged then Z₁ isnegatively charged, and if X₁ is negatively charged, then Z₁ ispositively charged; or a pharmaceutically acceptable salt thereof. 2.The compound of claim 1 further defined as:

wherein: Y₁ is alkanediyl_((C≦12)), alkenediyl_((C≦12)),arenediyl_((C≦12)), heteroarenediyl_((C≦12)),heterocycloalkanediyl_((C≦12)),-alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12)),-alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),-alkane-diyl_((C≦8))-heteroarenediyl_((C≦12)),-alkanediyl_((C≦8))-heteroarene-diyl_((C≦12))-alkanediyl_((C≦8)), or asubstituted version of any of these groups; A is —NR_(a)—, —S—, or —O—;wherein: R_(a) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)),or R_(a) is taken together with either R₃ or R₄ and isalkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),alkylaminodiyl_((C≦8)), or a substituted version of any of these groups;R₁ is a group of the formula:

wherein: R₅, R₆, and R₂ are each independently hydrogen oralkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,-alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), -alkanediyl_((C≦6))-NR′R″, ora substituted version of any of these groups wherein: R′ and R″ are eachindependently hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or—Z₂A′R₇; wherein: Z₂ is alkanediyl_((C≦4)) or substitutedalkanediyl_((C≦4)); A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;  R_(b) ishydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and  R_(j) ishydrogen, halo, hydroxy, acyloxy_((C≦24)), or substitutedacyloxy_((C≦24)); R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or R₅, R₆, and R₂ areeach independently —Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦4)) orsubstituted alkanediyl_((C≦4)); A″ is —CHR_(k)—, —C(O)O—, or—C(O)NR_(l)—;  R_(l) is hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)); and  R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),or substituted acyloxy_((C≦24)); and R₈ is alkyl_((C6-24)), substitutedalkyl_((C6-24)), alkenyl_((C6-24)), substituted alkenyl_((C6-24)); q is1, 2, or 3; and r is 1, 2, 3, or 4; R₁ is a group of the formula:

wherein: Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a substituted versionof any of these groups; R₉, R₁₀, and R₁₁ are each independently selectedfrom hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or —Z₄A′″R₁₂;wherein: Z₄ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4)); A′″is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;  R_(l) is hydrogen,alkyl_((C≦6)), or substituted alkyl_((C≦6)); and  R_(k) is hydrogen,halo, hydroxy, acyloxy_((C≦24)), or substituted acyloxy_((C≦24)); andR₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)); and x and y are 1, 2, 3, or 4; R₃ and R₄are each independently hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)), or R₃ or R₄ are taken together with R_(a) and isalkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),alkylaminodiyl_((C≦8)), or a substituted version of any of these groups;and m, n, and p are each independently an integer selected from 0, 1, 2,3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.
 3. Thecompound of claim 1 further defined as:

wherein: Y₁ is alkanediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),-alkanediyl_((C≦8))-heterocycloalkanediyl_((C≦12)),-alkanediyl_((C≦8))-hetero-cycloalkanediyl_((C≦12))-alkanediyl_((C≦8)),or a substituted version of any of these groups; A is —NR_(a)— or —O—;wherein: R_(a) is hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)),or R_(a) is taken together with either R₃ or R₄ and isalkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),alkylaminodiyl_((C≦8)), or a substituted version of any of these groups;R₁ is a group of the formula:

wherein: R₅, R₆, and R₂ are each independently hydrogen oralkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,-alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), -alkanediyl_((C≦6))-NR′R″, ora substituted version of any of these groups wherein: R′ and R″ are eachindependently hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or—Z₂A′R₇; wherein: Z₂ is alkanediyl_((C≦4)) or substitutedalkanediyl_((C≦4)); A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;  R_(b) ishydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and  R_(j) ishydrogen, halo, hydroxy, acyloxy_((C≦24)), or substitutedacyloxy_((C≦24)); R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or R₅, R₆, and R₂ areeach independently —Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦4)) orsubstituted alkanediyl_((C≦4)); A″ is —CHR_(k)—, —C(O)O—, or—C(O)NR_(l)—;  R_(l) is hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)); and  R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),or substituted acyloxy_((C≦24)); and R₈ is alkyl_((C6-24)), substitutedalkyl_((C6-24)), alkenyl_((C6-24)), substituted alkenyl_((C6-24)); q is1, 2, or 3; and r is 1, 2, 3, or 4; R₁ is a group of the formula:

wherein: Y₂ is arenediyl_((C≦12)), heterocycloalkanediyl_((C≦12)),heteroarenediyl_((C≦12)), alkoxydiyl_((C≦12)), or a substituted versionof any of these groups; R₉, R₁₀, and R₁₁ are each independently selectedfrom hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or —Z₄A′″R₁₂;wherein: Z₄ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4)); A′″is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—;  R_(l) is hydrogen,alkyl_((C≦6)), or substituted alkyl_((C≦6)); and  R_(k) is hydrogen,halo, hydroxy, acyloxy_((C≦24)), or substituted acyloxy_((C≦24)); andR₁₂ is alkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)); and x and y are 1, 2, 3, or 4; R₃ and R₄are each independently hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)), or R₃ or R₄ are taken together with R_(a) and isalkanediyl_((C≦8)), alkenediyl_((C≦8)), alkoxydiyl_((C≦8)),alkylaminodiyl_((C≦8)), or a substituted version of any of these groups;and m, n, and p are each independently an integer selected from 0, 1, 2,3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.
 4. Thecompound of claim 1 further defined as:

wherein: R₁ is a group of the formula:

wherein: R₅, R₆, and R₂ are each independently hydrogen oralkyl_((C≦8)), -alkanediyl_((C≦6))-NH₂,-alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), -alkanediyl_((C≦6))-NR′R″, ora substituted version of any of these groups wherein: R′ and R″ are eachindependently hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)), or—Z₂A′R₇; wherein: Z₂ is alkanediyl_((C≦4)) or substitutedalkanediyl_((C≦4)); A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;  R_(b) ishydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and R_(j) ishydrogen, halo, hydroxy, acyloxy_((C≦24)), or substitutedacyloxy_((C≦24)); R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or R₅, R₆, and R₂ areeach independently —Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦4)) orsubstituted alkanediyl_((C≦4)); A″ is —CHR_(k)—, —C(O)O—, or—C(O)NR_(l)—;  R_(l) is hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)); and  R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),or substituted acyloxy_((C≦24)); and R₈ is alkyl_((C6-24)), substitutedalkyl_((C6-24)), alkenyl_((C6-24)), substituted alkenyl_((C6-24)); q is1, 2, or 3; and r is 1, 2, 3, or 4; R_(a), R₃, and R₄ are eachindependently hydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); andm, n, and p are each independently an integer selected from 0, 1, 2, 3,4, 5, or 6; or a pharmaceutically acceptable salt thereof. 5.-7.(canceled)
 8. The compound of claim 1, wherein R_(a) is hydrogen, R₃ isalkyl_((C≦8)) or substituted alkyl_((C≦8)), or R₄ is alkyl_((C≦8)) orsubstituted alkyl_((C≦8)). 9-10. (canceled)
 11. The compound of claim 1,wherein m is 1 or 2, n is 2 or 3, or p is 1, 2, or
 3. 12.-13. (canceled)14. The compound of claim 1, wherein R₁ is a group of the formula:

wherein: R₅, R₆, and R₂ are each independently hydrogen oralkyl_((C≦8)), alkanediyl_((C≦6))-NH₂,-alkanediyl_((C≦6))-alkylamino_((C≦8)),-alkanediyl_((C≦6))-dialkylamino_((C≦12)), -alkanediyl_((C≦6))NR′R″, ora substituted version of any of these groups wherein: R′ and R″ are eachindependently hydrogen, alkyl_((C≦8)), substituted alkyl_((C≦8)),—Z₂A′R₇; wherein: Z₂ is alkanediyl_((C≦4)) or substitutedalkanediyl_((C≦4)); A′ is —CHR_(j)—, —C(O)O—, or —C(O)NR_(b)—;  R_(b) ishydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and R_(j) ishydrogen, halo, hydroxy, acyloxy_((C≦24)), or substitutedacyloxy_((C≦24)); R₇ is alkyl_((C6-24)), substituted alkyl_((C6-24)),alkenyl_((C6-24)), substituted alkenyl_((C6-24)); or R₅, R₆, and X₁ areeach independently —Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦4)) orsubstituted alkanediyl_((C≦4)); A″ is —CHR_(k)—, —C(O)O—, or—C(O)NR_(l)—; R_(l) is hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)); and R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),or substituted acyloxy_((C≦24)); and R₈ is alkyl_((C6-24)), substitutedalkyl_((C6-24)), alkenyl_((C6-24)), substituted alkenyl_((C6-24)); q is1, 2, or 3; and r is 1, 2, 3, or
 4. 15. The compound of claim 14,wherein r is 1 or
 2. 16. (canceled)
 17. The compound of claim 1, whereinR₅ is —Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦4)) or substitutedalkanediyl_((C≦4)); A″ is —CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—; R_(l) ishydrogen, alkyl_((C≦6)), or substituted alkyl_((C≦6)); and R_(k) ishydrogen, halo, hydroxy, acyloxy_((C≦24)), or substitutedacyloxy_((C≦24)); and R₈ is alkyl_((C6-24)), substitutedalkyl_((C6-24)), alkenyl_((C6-24)), substituted alkenyl_((C6-24)). 18.(canceled)
 19. The compound of claim 1, wherein R₆ is —Z₃A″R₈; wherein:Z₃ is alkanediyl_((C≦4)) or substituted alkanediyl_((C≦4)); A″ is—CHR_(k)—, —C(O)O—, or —C(O)NR_(l)—; R_(l) is hydrogen, alkyl_((C≦6)),or substituted alkyl_((C≦6)); and R_(k) is hydrogen, halo, hydroxy,acyloxy_((C≦24)), or substituted acyloxy_((C≦24)); and R₈ isalkyl_((C6-24)), substituted alkyl_((C6-24)), alkenyl_((C6-24)),substituted alkenyl_((C6-24)). 20.-22. (canceled)
 23. The compound ofclaim 1, wherein R₂ is —Z₃A″R₈; wherein: Z₃ is alkanediyl_((C≦4)) orsubstituted alkanediyl_((C≦4)); A″ is —CHR_(k)—, —C(O)O—, or—C(O)NR_(l)—; R_(l) is hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)); and R_(k) is hydrogen, halo, hydroxy, acyloxy_((C≦24)),or substituted acyloxy_((C≦24)); and R₈ is alkyl_((C6-24)), substitutedalkyl_((C6-24)), alkenyl_((C6-24)), substituted alkenyl_((C6-24)). 24.The compound of claim 1, wherein Z₃ is alkanediyl_((C1-2)). 25.(canceled)
 26. The compound of claim 1, wherein A″ is —CHR_(k)— andR_(k) is hydroxy. 27.-29. (canceled)
 30. The compound of claim 1,wherein R₈ is alkyl_((C6-24)) or substituted alkyl_((C6-24)). 31.-47.(canceled)
 48. A compound of the formula:

wherein: R₁, R₂, and R₃ are each independently hydrogen, alkyl_((C≦6)),substituted alkyl_((C≦6)), or a group of the formula:

wherein: R₇ and R₈ are each independently hydrogen, alkyl_((C≦6)),substituted alkyl_((C≦6)), or a group of the formula:

wherein: R₉ is hydrogen, halo, or hydroxy, or alkoxy_((C≦8)),acyloxy_((C≦8)), or a substituted version of either of these groups; andR₁₀ is alkyl_((C≦24)), alkenyl_((C≦24)), or a substituted version ofeither group; q is 1, 2, or 3; and r is 0, 1, 2, 3, or 4; R₄, R₅, and R₆are each independently hydrogen, alkyl_((C≦6)), or substitutedalkyl_((C≦6)), or R₄ is taken together with either R₅ or R₆ and isalkanediyl_((C≦2)), alkoxydiyl_((C≦2)), alkylaminodiyl_((C≦12)), or asubstituted version of any of these groups; and m and n are eachindependently 1, 2, 3, 4, or 5; or a pharmaceutically acceptable saltthereof. 49.-80. (canceled)
 81. A composition comprising: (A) a compoundof claim 1; and (B) a nucleic acid.
 82. The composition of claim 81,wherein the nucleic acid is a therapeutic nucleic acid. 83.-86.(canceled)
 87. The composition of claim 81, wherein the compositionfurther comprises a steroid or steroid derivative, a phospholipid, or aPEG lipid. 88.-95. (canceled)
 96. The composition of claim 81 furthercomprising a pharmaceutically acceptable carrier. 97-98. (canceled) 99.A method of treating a disease or disorder in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of a composition of claim
 81. 100.-110. (canceled)